Protease stabilized, acylated insulin analogues

ABSTRACT

Novel acylated insulin analogues exhibiting resistance towards proteases can, effectively, be administered pulmonary or orally. The insulin analogues contain B25H and A14E or A14H.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/922,117, filed Sep. 10, 2010, which is a 371 National Stage Filing ofInternational Application No. PCT/EP2009/053017, filed Mar. 13, 2009,which claimed priority of European Patent Application 08102708.8, filedMar. 18, 2008, and European Patent Application 08170231.8, filed Nov.28, 2008; this application also claims priority under 35 U.S.C. §119(e)of U.S. Provisional Application 61/039,120, filed Mar. 25, 2008; thecontents of all above-named applications are incorporated herein byreference.

FIELD OF THIS INVENTION

The present invention relates to novel acylated insulin analoguesexhibiting resistance towards proteases, a method for the preparation ofsuch insulin analogues, insulin preparations containing the insulinanalogues of the invention and a method of treating diabetes mellitususing these insulin analogues.

BACKGROUND OF THIS INVENTION

Diabetes mellitus is a metabolic disorder in which the ability toutilize glucose is partly or completely lost. About 5% of all peoplesuffer from diabetes and the disorder approaches epidemic proportions.Since the introduction of insulin in the 1920's, continuous efforts havebeen made to improve the treatment of diabetes mellitus. Since peoplesuffering from diabetes are subject to chronic treatment over severaldecades, there is a major need for safe, convenient and life qualityimproving insulin formulations.

The oral route is by far the most widely used route for drugadministration and is in general very well accepted by patients,especially for chronic therapies. Administration of therapeutic peptidesor proteins is however often limited to parenteral routes rather thanthe preferred oral administration due to several barriers such asenzymatic degradation in the gastrointestinal (GI) tract and intestinalmucosa, drug efflux pumps, insufficient and variable absorption from theintestinal mucosa, as well as first pass metabolism in the liver.

Normally, insulin formulations are administered by subcutaneousinjection. However, administration by other routes, e.g., orally orpulmonary, would be advantageous due to patient compliance, safety andconvenience. Some of the commercial available insulin formulations arecharacterized by a fast onset of action and other formulations have arelatively slow onset but show a more or less prolonged action. It isvary important for diabetic patients that there is, on the market, a bigvariety of insulins with different durations of actions (profiles ofactions). Briefly, insulins can be classified as being short-,intermediate- or long-acting.

WO 2008/034881 relates to certain insulin analogues wherein at least twohydrophobic amino acids have been substituted with hydrophilic aminoacids which insulin analogues are not acylated.

EP 2008/060733 and EP 2008/060733 relate to certain acylated insulinanalogues wherein the insulin analogue comprises an elongation with anamino acid or a peptide residue connected C terminally to the A21 aminoacid.

EP 2008/060734 relates to certain acylated insulins wherein an acylmoiety is attached to the parent insulin and wherein said acyl moietycomprises repeating units of alkylene glycol containing amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the oral effect of the compound of Example 183 on overnightfasted male Wistar rats.

FIGS. 2 a and 2 b show the oral effect of the compound of Example 9 onovernight fasted male Wistar rats.

FIG. 3 shows the oral effect of the compound of Example 16 on overnightfasted male Wistar rats.

FIG. 4 shows the oral effect of the compound of Example 25 on overnightfasted male Wistar rats.

FIG. 5 shows the oral effect of the compound of Example 27 on overnightfasted male Wistar rats.

FIG. 6 shows the oral effect of the compound of Example 24 on overnightfasted male Wistar rats.

FIG. 7 shows the oral effect of the compound of Example 13 on overnightfasted male Wistar rats.

FIG. 8 shows blood glucose lowering effects from intratracheal dropinstillation of the compound of Example 9, compared with a similar, butnon-protease resistant compound of Example 183.

FIG. 9 shows plasma insulin concentrations from intratracheal dropinstillation of the compound of Example 9, compared with a similar, butnon-protease resistant compound of Example 183.

FIGS. 10 and 11 show the pharmacokinetic profile of the compound ofExample 9 compared to the same compound but without the proteasestabilising A14E and 825H mutations. The data are from the sameexperiment. FIG. 10 is shown with the data from the first 250 minutes;FIG. 11 is shown with the full 24 hour (1440 minutes) time-course.

ASPECTS OF THIS INVENTION

An aspect of this invention relates to the furnishing of insulinanalogues which, when administered orally, can give a satisfactorycontrol of the blood glucose level.

Another aspect of this invention relates to furnishing of insulinanalogues which, when administered orally, can give a prolonged loweringof the glucose level

Another aspect of this invention relates to furnishing of basal insulinanalogues which, when administered orally, can give a prolonged loweringof the glucose level Another aspect of this invention relates tofurnishing of basal insulin analogues which, when administered orally,can give a satisfactory control of the blood glucose level followingthrice daily administration.

Another aspect of this invention relates to furnishing of basal insulinanalogues which, when administered orally, can give a satisfactorycontrol of the blood glucose level following twice daily administration.

Another aspect of this invention relates to furnishing of basal insulinanalogues which, when administered orally, can give a satisfactorycontrol of the blood glucose level following once daily administration.

Another aspect of this invention relates to furnishing of basal insulinanalogues which are hydrophilic.

Another aspect of this invention relates to furnishing of basal insulinanalogues which are more hydrophilic than human insulin.

Another aspect of this invention relates to furnishing of basal insulinanalogues which are less hydrophobic than human insulin, as measured bythe relative hydrophobicity (k′rel) as described herein.

Another aspect of this invention relates to furnishing of basal insulinanalogues which are less hydrophobic than of similar non-proteasestabilised parent insulins acylated with the same acyl moiety, asmeasured by the relative hydrophobicity (k′rel) as described herein.K′rel of the basal insulin analogues of the invention are preferablyless than 5, more preferably less than 3, more preferably less than 2,more preferably less than 1, more preferably less than 0.8, morepreferably less than 0.6, more preferably less than 0.5, more preferablyless than 0.4, more preferably less than 0.3, more preferably less than0.2, more preferably less than 0.1.

Another aspect of this invention relates to furnishing of basal insulinanalogues which, when administered orally, have satisfactorybioavailabilities. Compared with the bioavailabilities of similaracylated insulins without the protease stabilising mutations given insimilar doses, the bioavailability of preferred compounds of thisinvention is at least 10% higher, preferably 20% higher, preferably 25%higher, preferably 30% higher, preferably 35% higher, preferably 40%higher, preferably 45% higher, preferably 50% higher, preferably 55%higher, preferably 60% higher, preferably 65% higher, preferably 70%higher, preferably 80% higher, preferably 90% higher, preferably 100%higher, preferably more than 100% higher than that of the non-proteasestabilised comparator.

Another aspect of this invention relates to furnishing of basal insulinanalogues which, when administered orally, have satisfactorybioavailabilities. Bioavailabilities of preferred compounds of thisinvention (relative to i.v. administration) are at least 0.3%,preferably >0.5%, preferably >1%, preferably >1.5%, preferably >2%,preferably >2.5%, preferably >3%, preferably >3.5%, preferably >4%,preferably >5%, preferably >6%, preferably >7%, preferably >8%,preferably >9%, preferably >10%.

Another aspect of this invention relates to furnishing of basal insulinanalogues which, when administered by intravenous infusion, havesatisfactory potencies. Compared with the potency of human insulin,potencies of preferred protease stabilised insulin analogues of theinvention are preferably >5%, preferably >10%, preferably >20%,preferably >30%, preferably >40%, preferably >50%, preferably >75% andpreferably >100%.

Another aspect of this invention relates to the furnishing of insulinanalogues which, when administered pulmonarily, can give a satisfactorycontrol of the blood glucose level.

Another aspect of this invention relates to the furnishing of insulinanalogues which, when administered pulmonarily, can give a satisfactorycontrol of the blood glucose level with a relatively slow onset ofaction and/or a more or less prolonged action.

Another aspect of this invention relates to the furnishing of insulinanalogues having a satisfactory prolonged action following pulmonaryadministration. Compared with similar acylated insulin without proteasestabilising mutations given in similar doses, the duration of action ofpreferred compounds of this invention is at least 10% longer, preferably20% longer, preferably 25% longer, preferably 30% longer, preferably 35%longer, preferably 40% longer, preferably 45% longer, preferably 50%longer, preferably 55% longer, preferably 60% longer, preferably 65%longer, preferably 70% longer, preferably 80% longer, preferably 90%longer, preferably 100% longer, preferably more than 100% longer thanthat of the comparator. Duration of action can be measured by the timethat blood glucose is suppressed, or by measuring relevantpharmacokinetic properties, for example t_(1/2) or MRT (mean residencetime).

Another aspect of this invention relates to the furnishing of insulinanalogues having a satisfactory pulmonary bioavailability. Compared withthe bioavailability of human insulin or compared with similar acylatedinsulin without protease stabilising mutations given in similar doses,the bioavailability of preferred compounds of this invention is at least10% higher, preferably 20% higher, preferably 25% higher, preferably 30%higher, preferably 35% higher, preferably 40% higher, preferably 45%higher, preferably 50% higher, preferably 55% higher, preferably 60%higher, preferably 65% higher, preferably 70% higher, preferably 80%higher, preferably 90% higher, preferably 100% higher, preferably morethan 100% higher than that of the comparator.

Another aspect of this invention relates to the furnishing of insulinanalogues having increased apparent in vivo potency.

Another aspect of this invention relates to the furnishing of prolongedacting insulins with oral bioavailability.

Another aspect of this invention relates to the furnishing of insulinanalogues having an increased proteolytical stability compared to thestability of human insulin. Compared with human insulin, theproteolytical stability of preferred compounds of this invention is atleast 2 fold more stable, preferably 3 fold more stable, preferably 4fold more stable, preferably 5 fold more stable, preferably 6 fold morestable, preferably 7 fold more stable, preferably 8 fold more stable,preferably 9 fold more stable, preferably 10 fold more stable,preferably 12 fold more stable, preferably 14 fold more stable,preferably 16 fold more stable, preferably 18 fold more stable,preferably 20 fold more stable, preferably 25 fold more stable,preferably more than 25 fold more stable than that of the comparator.Proteolytical stability can be measured by exposing the insulins to (amixture of) proteolytic enzymes, e.g. an extract of gut enzymes asdescribed herein.

The object of this invention is to overcome or ameliorate at least oneof the disadvantages of the prior art, or to provide a usefulalternative.

DEFINITIONS

Herein, the term insulin covers natural occurring insulins, e.g., humaninsulin, as well as insulin analogues thereof. Human insulin consists oftwo polypeptide chains, the so-called A and B chains which contain 21and 30 amino acid residues, respectively, and which are interconnectedby two cystine disulphide bridges.

Herein, the term amino acid residue covers an amino acid from which ahydrogen atom has been removed from an amino group and/or a hydroxygroup has been removed from a carboxy group and/or a hydrogen atom hasbeen removed from a mercapto group. Imprecise, an amino acid residue maybe designated an amino acid.

Herein, hydrophobic amino acids are to be understood as the naturallyoccurring amino acids tryptophan (Trp, W), phenylalanine (Phe, F),valine (Val, V), isoleucine (Ile, I), leucine (Leu, L) and tyrosine(Tyr, Y) (with the three-letter and the one-letter abbreviation inbrackets).

Herein, hydrophilic amino acids are to be understood as natural aminoacids that are not hydrophobic amino acids according to the definitionabove. In one embodiment hydrophilic acids according to the inventionare selected from the group consisting of: Glutamic acid (Glu, E),aspartic acid (Asp, D), histidine (His, H), glutamine (Gln, Q),asparagine (Asn, N), serine (Ser, S), threonine (Thr, T), proline (Pro,P), glycine (Gly, G), lysine (Lys, K) and arginine (Arg, R). In afurther embodiment hydrophilic amino acids according to the inventionare selected from the group consisting of: Glutamic acid (Glu, E),aspartic acid (Asp, D), histidine (His, H), glutamine (Gln, Q),asparagine (Asn, N), lysine (Lys, K) and arginine (Arg, R).

Herein, the term insulin analogue covers a polypeptide which has amolecular structure which formally can be derived from the structure ofa naturally occurring insulin, e.g., human insulin, by deleting and/orsubstituting (replacing) one or more amino acid residue occurring in thenatural insulin and/or by adding one or more amino acid residue. Theadded and/or substituted amino acid residues can either be codable aminoacid residues or other naturally occurring amino acid residues or purelysynthetic amino acid residues. In a preferred embodiment, the insulinanalogue has two or more mutations compared to human insulin.

Herein, the term protease stabilised insulin means the insulin withoutan appended acyl moiety. Said protease stabilised insulins have animproved stability against degradation from proteases.

Herein, the term parent insulin means the insulin without an appendedacyl moiety and without mutations to improve stability againstdegradation from proteases. Said parent insulins have optionallymutations relative to human insulin. Parent insulins are thus alsoinsulin analogues as defined above. Herein, the terms parent insulin andnon-protease stabilised insulin covers the same compounds.

Herein, the term mutation covers any change in amino acid sequence(substitutions and insertions with codable amino acids as well asdeletions).

Herein, the term analogues of the A chain and analogues of the B chainsof human insulin covers A and B chains of human insulin, respectively,having one or more substitutions, deletions and or extensions(additions) of the A and B amino acid chains, respectively, relative tothe A and B chains, respectively, of human insulin.

Herein, terms like A1, A2, A3 etc. indicate the position 1, 2 and 3,respectively, in the A chain of insulin (counted from the N-terminalend). Similarly, terms like B1, B2, B3 etc. indicates the position 1, 2and 3, respectively, in the B chain of insulin (counted from theN-terminal end). Using the one letter codes for amino acids, terms likeA21A, A21G and A21Q designates that the amino acid in the A21 positionis A, G and Q, respectively. Using the three letter codes for aminoacids, the corresponding expressions are AlaA21, GlyA21 and GlnA21,respectively.

Herein, the terms A(0) or B(0) indicate the positions N-terminallyneighbouring the A1 or B1 positions, respectively, in the A or B chains,respectively. The terms A(−1) or B(−1) indicate the positions of thefirst amino acids N-terminally to A(0) or B(0), respectively. Thus A(−2)and B(−2) indicate positions N-terminally to A(−1) and B(−1),respectively, A(−3) and B(−3) indicate positions N-terminally to A(−2)and B(−2), respectively, and so forth.

Herein, terms like desB29 and desB30 indicate an insulin analoguelacking the B29 or B30 amino acid residue, respectively.

Herein, the term “fast acting insulin” covers an insulin having a fasteronset of action than normal or regular human insulin.

Herein, the term “long acting insulin” or the term “basal insulin”covers an insulin having a longer duration of action than normal orregular human insulin. Preferably, the time-action is more than 5, or 8hours, in particularly of at least 9 hours. Preferably, the basalinsulin has a time-action of at least 10 hours. The basal insulin maythus have a time-action in the range from about 8 to 24 hours,preferably in the range from about 9 to about 15 hours.

The numbering of the positions in insulin analogues, insulins and A andB chains is done so that the parent compound is human insulin with thenumbering used for it.

Herein, the term “acylated insulin” covers modification of insulin byattachment of one or more acyl moieties via a linker to the proteasestabilised insulin.

By acylated insulin having insulin activity is meant an acylated insulinwith either the ability to lower the blood glucose in mammalians asmeasured in a suitable animal model, which may, e.g., be a rat, rabbit,or pig model, after suitable administration, e.g., by intravenous orsubcutaneous administration, or an insulin receptor binding affinity.

Herein, the term alkyl covers a saturated, branched or straighthydrocarbon group.

Herein, the term alkoxy covers the radical “alkyl-O—”. Representativeexamples are methoxy, ethoxy, propoxy (e.g., 1-propoxy and 2-propoxy),butoxy (e.g., 1-butoxy, 2-butoxy and 2-methyl-2-propoxy), pentoxy(1-pentoxy and 2-pentoxy), hexoxy (1-hexoxy and 3-hexoxy), and the like.

Herein, the term alkylene covers a saturated, branched or straightbivalent hydrocarbon group having from 1 to 12 carbon atoms.Representative examples include, but are not limited to, methylene;1,2-ethylene; 1,3-propylene; 1,2-propylene; 1,3-butylene; 1,4-butylene;1,4-pentylene; 1,5-pentylene; 1,5-hexylene; 1,6-hexylene; and the like.

Herein, the term “neutral linear amino acid” covers. Non limitingexamples of neutral linear amino acids are.

Herein, the term “cyclic amino acid” covers. Non limiting examples ofcyclic amino acids are.

Herein, the term “acidic amino acid” covers. Non limiting examples ofacidic amino acids are.

Herein, the term “fatty acid” covers a linear or branched, aliphaticcarboxylic acids having at least two carbon atoms and being saturated orunsaturated. Non limiting examples of fatty acids are myristic acid,palmitic acid, and stearic acid.

Herein, the term “fatty diacid” covers a linear or branched, aliphaticdicarboxylic acids having at least two carbon atoms and being saturatedor unsaturated. Non limiting examples of fatty diacids are succinicacid, hexanedioic acid, octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,heptadecanedioic acid, octadecanedioic acid, and eicosanedioic acid.

Herein, the naming of the insulins is done according to the followingprinciples: The names are given as mutations and modifications(acylations) relative to human insulin. For the naming of the acylmoiety, the naming is done according to IUPAC nomenclature and in othercases as peptide nomenclature. For example, naming the acyl moiety:

can for example be “octadecanedioyl-γGlu-OEG-OEG”, or“17-carboxyheptadecanoyl-γGlu-OEG-OEG”, wherein

OEG is short hand notation for the amino acid NH₂(CH₂)₂O(CH₂)₂OCH₂CO₂H,

γGlu is short hand notation for the amino acid gamma glutamic acid.

Other short hand notations for amino acids are, for example:

PEG3 is NH₂((CH₂)₂O)₄CH₂CH₂CO₂H

PEG7 is NH₂((CH₂)₂O)₈CH₂CH₂CO₂H

For example, the insulin of example 9 (with the sequence/structure givenbelow) is named “A14E, B25H, B29K (N^(∈)Octadecanedioyl-γGlu-OEG-OEG),desB30 human insulin” to indicate that the amino acid in position A14, Yin human insulin, has been mutated to E, the amino acid in position B25,F in human insulin, has been mutated to H, the amino acid in positionB29, K as in human insulin, has been modified by acylation on theepsilon nitrogen in the lysine residue of B29, denoted N^(∈), by theresidue octadecanedioyl-γGlu-OEG-OEG, and the amino acid in positionB30, T in human insulin, has been deleted. Asterisks in the formulabelow indicate that the residue in question is different (i.e. mutated)as compared to human insulin. Throughout this application both formulasand names of preferred insulins of the invention are given

Herein, the term “chemical stability” and “high chemical stability”,means that chemically, the insulins of the invention are sufficientlystable in the desired formulation. That is that chemical degradationproducts are only formed in amounts that do not compromise shelf life ofthe final drug product. Chemical degradation products includesdeamidation products, iso-aspartate formation, dimer formation,racemisation products, products resulting from dehydration processesetcetera. Chemical stability may be measured by HPLC analyses of agedsamples or formulations.

Herein, the term “high physical stability” covers a tendency tofibrillation being less than 50% of that of human insulin. Fibrillationmay be described by the lag time before fibril formation is initiated ata given conditions.

A polypeptide with insulin receptor and IGF-1 receptor affinity is apolypeptide which is capable of interacting with an insulin receptor anda human IGF-1 receptor in a suitable binding assay. Such receptor assaysare well-know within the field and are further described in theexamples. The present acylated insulin will not bind to the IGF-1receptor or will have a rather low affinity to said receptor. Moreprecisely, the acylated insulins of this invention will have an affinitytowards the IGF-1 receptor of substantially the same magnitude or lessas that of human insulin

The term “pharmaceutically acceptable” as used herein means suited fornormal pharmaceutical applications, i.e., giving rise to no seriousadverse events in patients etc.

The terms treatment and treating as used herein means the management andcare of a patient for the purpose of combating a disease, disorder orcondition. The term is intended to include the delaying of theprogression of the disease, disorder or condition, the alleviation orrelief of symptoms and complications, and/or the cure or elimination ofthe disease, disorder or condition. The patient to be treated ispreferably a mammal, in particular a human being.

The term treatment of a disease as used herein means the management andcare of a patient having developed the disease, condition or disorder.The purpose of treatment is to combat the disease, condition ordisorder. Treatment includes the administration of the active compoundsto eliminate or control the disease, condition or disorder as well as toalleviate the symptoms or complications associated with the disease,condition or disorder.

The term prevention of a disease as used herein is defined as themanagement and care of an individual at risk of developing the diseaseprior to the clinical onset of the disease. The purpose of prevention isto combat the development of the disease, condition or disorder, andincludes the administration of the active compounds to prevent or delaythe onset of the symptoms or complications and to prevent or delay thedevelopment of related diseases, conditions or disorders.

The term effective amount as used herein means a dosage which issufficient in order for the treatment of the patient to be effectivecompared with no treatment.

POT is the Schizosaccharomyces pombe triose phosphate isomerase gene,and TPI1 is the S. cerevisiae triose phosphate isomerase gene.

By a leader is meant an amino acid sequence consisting of a pre-peptide(the signal peptide) and a pro-peptide.

The term signal peptide is understood to mean a pre-peptide which ispresent as an N-terminal sequence on the precursor form of a protein.The function of the signal peptide is to allow the heterologous proteinto facilitate translocation into the endoplasmic reticulum. The signalpeptide is normally cleaved off in the course of this process. Thesignal peptide may be heterologous or homologous to the yeast organismproducing the protein. A number of signal peptides which may be usedwith the DNA construct of this invention including yeast asparticprotease 3 (YAP3) signal peptide or any functional analog (Egel-Mitaniet al. (1990) YEAST 6:127-137 and U.S. Pat. No. 5,726,038) and theα-factor signal of the MFα1 gene (Thorner (1981) in The MolecularBiology of the Yeast Saccharomyces cerevisiae, Strathern et al., eds.,pp 143-180, Cold Spring Harbor Laboratory, NY and U.S. Pat. No.4,870,00.

Herein, the term “pro-peptide” covers a polypeptide sequence whosefunction is to allow the expressed polypeptide to be directed from theendoplasmic reticulum to the Golgi apparatus and further to a secretoryvesicle for secretion into the culture medium (i.e. exportation of thepolypeptide across the cell wall or at least through the cellularmembrane into the periplasmic space of the yeast cell). The pro-peptidemay be the yeast a-factor pro-peptide, vide U.S. Pat. Nos. 4,546,082 and4,870,008. Alternatively, the pro-peptide may be a syntheticpro-peptide, which is to say a pro-peptide not found in nature. Suitablesynthetic pro-peptides are those disclosed in U.S. Pat. Nos. 5,395,922;5,795,746; 5,162,498 and WO 98/32867. The pro-peptide will preferablycontain an endopeptidase processing site at the C-terminal end, such asa Lys-Arg sequence or any functional analogue thereof.

Unless indicated explicitly, the amino acids mentioned herein areL-amino acids. Further, the left and right ends of an amino acidsequence of a peptide are, respectively, the N- and C-termini, unlessotherwise specified.

SUMMARY OF THE INVENTION

It has been discovered that insulins that are stabilised towardsproteolytic degradation (by specific mutations) and acylated at theB29-lysine are efficacious and protracted and possess high potential asprotracted insulins that can be administered pulmonary or orally. Theacylation confers binding to serum albumin, and, consequently,protraction. In addition, the acylated insulins of the invention displaysubstantial reduction of insulin receptor affinity, compared to similaracylated insulins that are not stabilised towards proteolyticdegradation. This reduction in insulin receptor affinity ofalbumin-bound insulins of the invention contributes to the protractionof the acylated insulin in circulation, since insulin is internalisedand degraded upon receptor activation. Hence, clearance of the insulinsof the invention is reduced. The reduction of insulin receptor affinitydoes probably not cause a loss of potency, e.g., as measured in thehyperinsulinaemic euglycaemic clamp as described herein. The combinationof high albumin binding affinity and low insulin receptor affinity is,thus, beneficial for obtaining long duration of action of the insulins(basal insulins). Furthermore, after oral administration, these acylatedinsulins have a higher degree of bioavailability than similar knownacylated insulins, that are not stabilised towards proteolyticdegradation. Hence, these acylated insulin analogues are valuable fororal administration. Similarly, after pulmonary administration, theseacylated protease stabilised insulins displays higher apparent potencyand/or bioavailability than similar known acylated insulins, that arenot stabilised towards proteolytic degradation. Furthermore, theseacylated protease stabilised insulins displays protracted time-actionprofiles when administered pulmonary to mammals. Hence, these acylatedinsulin analogues are valuable for pulmonary administration.

The above-mentioned insulins that are stabilised towards proteolyticdegradation are herein designated protease stabilised insulins.

The protease stabilised insulin molecule has a limited number of thenaturally occurring amino acid residues substituted with other aminoacid residues relative to human insulin as explained in the detailedpart of the specification.

In one embodiment, this invention relates to an acylated insulin,wherein the protease stabilised insulin analogue deviates from humaninsulin in one or more of the following deletions or substitutions: Q inposition A18, A, G or Q in position A21, G or Q in position B1 or noamino acid residue in position B1, Q, S or T in position B3 or no aminoacid residue in position B3, Q in position B13, no amino acid residue inposition B27, D, E or R in position B28 and no amino acid in positionB30.

In still a further aspect, this invention relates to pharmaceuticalpreparations comprising the acylated insulin of this invention andsuitable adjuvants and additives such as one or more agents suitable forstabilization, preservation or isotoni, e.g., zinc ions, phenol, cresol,a parabene, sodium chloride, glycerol or mannitol. The zinc content ofthe present formulations may be between 0 and about 6 zinc atoms per 6molecules of insulin. The pH value of the pharmaceutical preparation maybe between about 4 and about 8.5, between about 4 and about 5 or betweenabout 6.5 and about 7.5.

In a further embodiment, this invention is related to the use of theacylated insulin as a pharmaceutical for the reducing of blood glucoselevels in mammalians, in particularly for the treatment of diabetes.

In a further aspect, this invention is related to the use of theacylated insulin for the preparation of a pharmaceutical preparation forthe reducing of blood glucose level in mammalians, in particularly forthe treatment of diabetes.

In a further embodiment, this invention is related to a method ofreducing the blood glucose level in mammalians by administrating atherapeutically active dose of an acylated insulin of this invention toa patient in need of such treatment.

In a further aspect of this invention, the acylated insulins areadministered in combination with one or more further active substancesin any suitable ratios. Such further active agents may be selected fromhuman insulin, fast acting insulin analogues, antidiabetic agents,antihyperlipidemic agents, antiobesity agents, antihypertensive agentsand agents for the treatment of complications resulting from orassociated with diabetes.

In one embodiment, the two active components are administered as a mixedpharmaceutical preparation. In another embodiment, the two componentsare administered separately either simultaneously or sequentially.

In one embodiment, the acylated insulins of this invention may beadministered together with fast acting human insulin or human insulinanalogues. Such fast acting insulin analogue may be such wherein theamino acid residue in position B28 is Asp, Lys, Leu, Val, or Ala and theamino acid residue in position B29 is Lys or Pro, des(B28-B30) humaninsulin, des(B27) human insulin or des(B30) human insulin, and ananalogue wherein the amino acid residue in position B3 is Lys and theamino acid residue in position B29 is Glu or Asp. The acylated insulinof this invention and the rapid acting human insulin or human insulinanalogue can be mixed in a ratio from about 90% of the acylated insulinto about 10% of the rapid acting human insulin or human insulinanalogue; preferably from about 70% of the acylated insulin to about 30%of the rapid acting human insulin or human insulin analogue, and evenmore preferred from about 50% of the acylated insulin to about 50% ofthe rapid acting human insulin or human insulin analogue (% being weightpercentage).

The acylated insulins of this invention may also be used on combinationtreatment together with an antidiabetic agent.

Antidiabetic agents will include insulin, GLP-1 (1-37) (glucagon likepeptide-1) described in WO 98/08871, WO 99/43706, U.S. Pat. No.5,424,286, WO 00/09666, WO 2006/097537, PCT/EP2008/061755 andPCT/EP2008/061830, GLP-2, exendin-4(1-39), insulinotropic fragmentsthereof, insulinotropic analogues thereof and insulinotropic derivativesthereof. Insulinotropic fragments of GLP-1(1-37) are insulinotropicpeptides for which the entire sequence can be found in the sequence ofGLP-1(1-37) and where at least one terminal amino acid has been deleted.

The acylated insulins of this invention may also be used on combinationtreatment together with an oral antidiabetic such as a thiazolidindione,metformin and other type 2 diabetic pharmaceutical preparation for oraltreatment.

Furthermore, the acylated insulin of this invention may be administeredin combination with one or more antiobesity agents or appetiteregulating agents.

In one embodiment this invention is related to a pulmonal pharmaceuticalpreparation comprising the acylated insulin of this invention andsuitable adjuvants and additives such as one or more agents suitable forstabilization, preservation or isotoni, e.g., zinc ions, phenol, cresol,a parabene, sodium chloride, glycerol, propyleneglycol or mannitol.

It should be understood that any suitable combination of the acylatedinsulins with diet and/or exercise, one or more of the above-mentionedcompounds and optionally one or more other active substances areconsidered to be within the scope of this invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The stability and solubility properties of insulin are importantunderlying aspects for current insulin therapy. This invention isaddressed to these issues by providing stable, acylated insulinanalogues wherein the acylation decreases molecular flexibility andconcomitantly reduce the fibrillation propensity and limit or modify thepH precipitation zone.

The acylated insulins of this invention are in particularly intended forpulmonary or oral administration due to their relatively highbioavailability compared to, e.g., human insulin and acylated humaninsulin. Furthermore, the acylated insulins will have a protractedinsulin activity.

As mentioned above, insulins that are stabilised towards proteolyticdegradation are herein designated protease stabilised insulins. Theacylated insulins of this invention are said protease stabilisedinsulins which have been acylated as described herein.

Said protease stabilised insulins are derived from insulin compoundswhich herein are designated parent insulins or non-protease stabilisedinsulins.

In one embodiment a parent insulin is selected from the group consistingof a) human insulin; b) an insulin analogue of human insulin wherein theamino acid residue in position B28 of is Pro, Asp, Lys, Leu, Val, or Alaand the amino acid residue in position B29 is Lys or Pro and optionallythe amino acid residue in position B30 is deleted; c) an insulinanalogue which is des(B28-B30) human insulin, des(B27) human insulin ordes(B30) human insulin; d) an insulin analogue of human insulin whereinthe amino acid residue in position B3 is Lys and the amino acid residuein position B29 is Glu or Asp; e) an insulin analogue of human insulinwherein the amino acid residue in position A21 is Gly and wherein theinsulin analogue is further extended in the C-terminal with two arginineresidues; f) an insulin derivative wherein the amino acid residue inposition B30 is substituted with a threonine methyl ester; and g) aninsulin derivative wherein to the NE position of lysine in the positionB29 of des(B30) human insulin a tetradecanoyl chain is attached. Each ofthese groups is a specific embodiment.

In another embodiment, a parent insulin is selected from the groupconsisting of human insulin; desB30 human insulin; AspB28 human insulin;AspB28,DesB30 human insulin; LysB3,GluB29 human insulin; LysB28,ProB29human insulin; GlyA21, ArgB31, ArgB32 human insulin; and desB30, ArgB31,ArgB32 human insulin.

More specifically, the protease stabilised insulin is an insulinmolecule having two or more mutations of the A and/or B chain relativeto the parent insulin. Surprisingly, it has been found that bysubstituting two or more hydrophobic amino acids within or in closeproximity to two or more protease sites on an insulin with hydrophilicamino acids, an insulin analogue (i.e., a protease stabilised insulin)is obtained which is proteolytically more stable compared to the parentinsulin. In a broad aspect, a protease stabilised insulin is an insulinanalogue wherein at least two hydrophobic amino acids have beensubstituted with hydrophilic amino acids relative to the parent insulin,wherein the substitutions are within or in close proximity to two ormore protease cleavage sites of the parent insulin and wherein suchinsulin analogue optionally further comprises one or more additionalmutations.

In another embodiment, a protease stabilised insulin is an insulinanalogue wherein

-   -   the amino acid in position A12 is Glu or Asp and/or the amino        acid in position A13 is His, Asn, Glu or Asp and/or the amino        acid in position A14 is Asn, Gln, Glu, Arg, Asp, Gly or His        and/or the amino acid in position A15 is Glu or Asp; and    -   the amino acid in position B24 is His and/or the amino acid in        position B25 is His and/or the amino acid in position B26 is        His, Gly, Asp or Thr and/or the amino acid in position B27 is        His, Glu, Gly or Arg and/or the amino acid in position B28 is        His, Gly or Asp; and        which optionally further comprises one or more additional        mutations.

In another embodiment a protease stabilised insulin is an analoguecomprising the B25H or B25N mutations in combination with mutations inB27, optionally in combination with other mutations.

In another embodiment a protease stabilised insulin is an analoguecomprising the B25H or B25N mutations in combination with mutations inB27, optionally in combination with other mutations. The mutations inposition B27 can, for example, be Glu or Asp.

These protease stabilised acyated insulin analogues comprising both theB25 and B27 mutations have advantageous properties.

In another embodiment, a protease stabilised insulin is an insulinanalogue comprising an A-chain amino acid sequence of formula 1:

(SEQ ID No: 1)Xaa_(A(−2))-Xaa_(A(−1))-Xaa_(A0)-Gly-Ile-Val-Glu-Gln-Cys-Cys-Xaa_(A8)-Ser-Ile-Cys-Xaa_(A12)-Xaa_(A13)-Xaa_(A14)-Xaa_(A15)-Leu-Glu-Xaa_(A18)-Tyr-Cys-Xaa_(A21) Formula (1)and a B-chain amino acid sequence of formula 2:

(SEQ ID No: 2)Xaa_(B(−2))-Xaa_(B(−1))-Xaa_(B0)-Xaa_(B1)-Xaa_(B2)-Xaa_(B3)-Xaa_(B4)-His-Leu-Cys-Gly-Ser-XaaB10-Leu-Val-Glu-Ala-Leu-Xaa_(B16)-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa_(B24)-Xaa_(B25)-Xaa_(B26)-Xaa_(B27)-Xaa_(B28)-Xaa_(B29)-Xaa_(B30)-Xaa_(B31)-Xaa_(B32) Formula (2)

wherein

Xaa_(A(−2)) is absent or Gly;Xaa⁽⁻¹⁾ is absent or Pro;Xaa_(A0) is absent or Pro;Xaa_(A8) is independently selected from Thr and His;Xaa_(A12) is independently selected from Ser, Asp and Glu;Xaa_(A13) is independently selected from Leu, Thr, Asn, Asp, Gln, His,Lys, Gly, Arg, Pro, Ser and Glu;Xaa_(A14) is independently selected from Tyr, Thr, Asn, Asp, Gln, His,Lys, Gly, Arg, Pro, Ser and Glu;Xaa_(A15) is independently selected from Gln, Asp and Glu;Xaa_(A18) is independently selected from Asn, Lys and Gln;Xaa_(A21) is independently selected from Asn and Gln;Xaa_(B(−2)) is absent or Gly;Xaa_(B(−1)) is absent or Pro;Xaa_(B0) is absent or Pro;Xaa_(B1) is absent or independently selected from Phe and Glu;Xaa_(B2) is absent or Val;Xaa_(B3) is absent or independently selected from Asn and Gln;Xaa_(B4) is independently selected from Gln and Glu;Xaa_(B10) is independently selected from His, Asp, Pro and Glu;Xaa_(B16) is independently selected from Tyr, Asp, Gln, His, Arg, andGlu;Xaa_(B24) is independently selected from Phe and His;Xaa_(B25) is independently selected from Asn, Phe and His;Xaa_(B26) is absent or independently selected from Tyr, His, Thr, Glyand Asp;Xaa_(B27) is absent or independently selected from Thr, Asn, Asp, Gln,His, Lys, Gly, Arg, Pro, Ser and Glu;Xaa_(B28) is absent or independently selected from Pro, His, Gly andAsp;Xaa_(B29) is absent or independently selected from Lys, Arg and Gln;and, preferably, Xaa_(B29) is absent or independently selected from Lysand Gln;Xaa_(B30) is absent or Thr;Xaa_(B31) is absent or Leu;Xaa_(B32) is absent or Glu;

the C-terminal may optionally be derivatized as an amide;

wherein the A-chain amino acid sequence and the B-chain amino acidsequence are connected by disulphide bridges between the cysteines inposition 7 of the A-chain and the cysteine in position 7 of the B-chain,and between the cysteine in position 20 of the A-chain and the cysteinein position 19 of the B-chain and wherein the cysteines in position 6and 11 of the A-chain are connected by a disulphide bridge.

In another embodiment, a protease stabilised insulin is an insulinanalogue comprising an A-chain amino acid sequence of formula 3:

(SEQ ID No: 3)Gly-Ile-Val-Glu-Gln-Cys-Cys-Xaa_(A8)-Ser-Ile-Cys-Xaa_(A12)-Xaa_(A13)-Xaa_(A14)-Xaa_(A15)-Leu-Glu-Xaa_(A18)-Tyr-Cys-Xaa_(A21) Formula (3)and a B-chain amino acid sequence of formula 4:

(SEQ ID No: 4)Xaa_(B1)-Val-Xaa_(B3)-Xaa_(B4)-His-Leu-Cys-Gly-Ser-Xaa_(B10)-Leu-Val-Glu-Ala-Leu-Xaa_(B16)-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa_(B24)-His-Xaa_(B26)-Xaa_(B27)-Xaa_(B28)-Xaa_(B29)-Xaa_(B30)Formula (4) 

wherein

Xaa_(A8) is independently selected from Thr and His;Xaa_(A12) is independently selected from Ser, Asp and Glu;Xaa_(A13) is independently selected from Leu, Thr, Asn, Asp, Gln, His,Lys, Gly, Arg, Pro, Ser and Glu;Xaa_(A14) is independently selected from Thr, Asn, Asp, Gln, His, Lys,Gly, Arg, Pro, Ser and Glu;Xaa_(A18) is independently selected from Gln, Asp and Glu;Xaa_(A18) is independently selected from Asn, Lys and Gln;Xaa_(A21) is independently selected from Asn, and Gln;Xaa_(B1) is independently selected from Phe and Glu;Xaa_(B3) is independently selected from Asn and Gln;Xaa_(B4) is independently selected from Gln and Glu;Xaa_(B10) is independently selected from His, Asp, Pro and Glu;Xaa_(B18) is independently selected from Tyr, Asp, Gln, His, Arg, andGlu;Xaa_(B24) is independently selected from Phe and His;Xaa_(B26) is absent or independently selected from Tyr, His, Thr, Glyand Asp;Xaa_(B27) is absent or independently selected from Thr, Asn, Asp, Gln,His, Lys, Gly, Arg, Pro, Ser and Glu;Xaa_(B28) is absent or independently selected from Pro, His, Gly andAsp;Xaa_(B29) is absent or independently selected from Lys, Arg and Gln;and, preferably, Xaa_(B29) is absent or independently selected from Lysand Gln;Xaa_(B30) is absent or Thr;

the C-terminal may optionally be derivatized as an amide;

wherein the A-chain amino acid sequence and the B-chain amino acidsequence are connected by disulphide bridges between the cysteines inposition 7 of the A-chain and the cysteine in position 7 of the B-chain,and between the cysteine in position 20 of the A-chain and the cysteinein position 19 of the B-chain and wherein the cysteines in position 6and 11 of the A-chain are connected by a disulphide bridge.

In another embodiment, a protease stabilised insulin is an insulinanalogue wherein

Xaa_(A8) is independently selected from Thr and His;Xaa_(A12) is independently selected from Ser and Glu;Xaa_(A13) is independently selected from Leu, Thr, Asn, Asp, Gln, His,Lys, Gly, Arg, Pro, Ser and Glu;Xaa_(A14) is independently selected from Asp, His, and Glu;Xaa_(A15) is independently selected from Gln and Glu;Xaa_(A18) is independently selected from Asn, Lys and Gln;Xaa_(A21) is independently selected from Asn, and Gln;Xaa_(B1) is independently selected from Phe and Glu;Xaa_(B3) is independently selected from Asn and Gln;Xaa_(B4) is independently selected from Gln and Glu;Xaa_(B10) is independently selected from His, Asp, Pro and Glu;Xaa_(B16) is independently selected from Tyr, Asp, Gln, His, Arg, andGlu;Xaa_(B24) is independently selected from Phe and His;Xaa_(B25) is independently selected from Phe, Asn and His;Xaa_(B26) is independently selected from Tyr, Thr, Gly and Asp;Xaa_(B27) is independently selected from Thr, Asn, Asp, Gln, His, Lys,Gly, Arg, and Glu;Xaa_(B28) is independently selected from Pro, Gly and Asp;Xaa_(B29) is independently selected from Lys and Gln;Xaa_(B30) is absent or Thr;

the C-terminal may optionally be derivatized as an amide;

wherein the A-chain amino acid sequence and the B-chain amino acidsequence are connected by disulphide bridges between the cysteines inposition 7 of the A-chain and the cysteine in position 7 of the B-chain,and between the cysteine in position 20 of the A-chain and the cysteinein position 19 of the B-chain and wherein the cysteines in position 6and 11 of the A-chain are connected by a disulphide bridge.

Other embodiments of protease stabilised insulins are mentioned below.

A “protease” or a “protease enzyme” is a digestive enzyme which degradesproteins and peptides and which is found in various tissues of the humanbody such as e.g. the stomach (pepsin), the intestinal lumen(chymotrypsin, trypsin, elastase, carboxypeptidases, etc.) or mucosalsurfaces of the GI tract (aminopeptidases, carboxypeptidases,enteropeptidases, dipeptidyl peptidases, endopeptidases, etc.), theliver (Insulin degrading enzyme, cathepsin D etc), and in other tissues.

A proteolytically stable insulin analogue (also designated a proteasestabilised insulin) is herein to be understood as an insulin analogue,which is subjected to slower degradation by one or more proteasesrelative to human insulin. In one embodiment, a protease stabilisedinsulin is subjected to slower degradation by one or more proteasesrelative to the parent insulin. In a further embodiment, a proteasestabilised insulin is stabilized against degradation by one or moreenzymes selected from the group consisting of: pepsin (such as, e.g.,the isoforms pepsin A, pepsin B, pepsin C and/or pepsin F), chymotrypsin(such as, e.g., the isoforms chymotrypsin A, chymotrypsin B and/orchymotrypsin C), trypsin, Insulin-Degrading Enzyme (IDE), elastase (suchas, e.g., the isoforms pancreatic elastase I and/or II),carboxypeptidase (e.g., the isoforms carboxypeptidase A,carboxypeptidase A2 and/or carboxypeptidase B), aminopeptidase,cathepsin D and other enzymes present in intestinal extracts derivedfrom rat, pig or human.

In one embodiment, a protease stabilised insulin is stabilized againstdegradation by one or more enzymes selected from the group consistingof: chymotrypsin, trypsin, Insulin-Degrading Enzyme (IDE), elastase,carboxypeptidases, aminopeptidases and cathepsin D. In a furtherembodiment, a protease stabilised insulin is stabilized againstdegradation by one or more enzymes selected from the group consistingof: chymotrypsin, carboxypeptidases and IDE. In a yet furtherembodiment, a protease stabilised insulin is stabilized againstdegradation by one or more enzymes selected from: chymotrypsin andcarboxypeptidases.

T½ may be determined as described in the Examples as a measure of theproteolytical stability of a protease stabilised insulin towardsprotease enzymes such as chymotrypsin, pepsin and/or carboxypeptidase A.In one embodiment of the invention, T½ is increased relative to humaninsulin. In a further embodiment, T½ is increased relative to the parentinsulin. In a yet further embodiment, T½ is increased at least 2-foldrelative to the parent insulin. In a yet further embodiment, T½ isincreased at least 3-fold relative to the parent insulin. In a yetfurther embodiment, T½ is increased at least 4-fold relative to theparent insulin. In a yet further embodiment, T½ is increased at least5-fold relative to the parent insulin. In a yet further embodiment, T½is increased at least 10-fold relative to the parent insulin.

An alternative way of measuring proteolytical stability is to measurethe relative stability towards a comparator, e.g., human insulin. Therelative stability is defines as T½/T½ (comparator), where T½ andT½(compatator) are the half-lives of the analogue and the comparator,respectively, in the degradation assay. In the examples section, therelative stability of selected insulins of the invention towards anenzyme mixture extracted from duodemum from rats is given (relative tohuman insulin as well as relative to a protease-resistant insulinwithout acylation).

Protease cleavage sites (herein also mentioned as protease sites) are tobe understood as amino acid residues that are recognized by proteasesand/or amino acid residues whose peptide bond is cleaved by proteases.Protease cleavage sites may be determined by determining cleavage“hotspots” by HPLC, MS or LC-MS analyses and/or by prediction based onenzyme specificity of the protease enzyme for which the proteasecleavage site is to be determined. A skilled person in the art will knowhow to determine protease cleavage sites for example based on enzymespecificities as for example described in Handbook of ProteolyticalEnzymes, 2nd ed., Barrett, A. J., Rawlings, N. D., Woesner, J. F.editors, Elsevier Academic Press 2004. For example chymotrypsin ispredicted to cleave peptide bonds C-terminal to aromatic residues (Trp,Tyr, Phe or Leu), that are not followed by Pro. Similarly, trypsin ispredicted to cleave peptide bonds C-terminal to basic residues Lys orArg, that are not followed by Pro, elastase is predicted to cleaveresidues C-terminal to Ala, Val, Gly or Ser and carboxypeptidase A willremove any C-terminal amino acid, but not Arg, Lys or Pro.Insulin-degrading enzyme (IDE) is predicted to cleave the followingpositions of human insulin B9-10, B10-11, B13-14, B14-15, B24-25,B25-26, A13-14 and A14-15.

The term substituting (an) amino acid “within or in close proximity” toa protease cleavage site is herein used to indicate the substitution ofan amino acid within or in close proximity to a position of the parentinsulin which has been determined to be a protease cleavage site. In oneembodiment, two or more hydrophobic amino acids within or in closeproximity to two or more protease sites on an insulin are substituted,wherein said hydrophobic amino acids are substituted with hydrophilicamino acids. In a further embodiment, two or more hydrophobic aminoacids within two or more protease sites on an insulin are substitutedwith hydrophilic amino acids. In a yet further embodiment, two or morehydrophobic amino acids situated next to two or more protease sites onan insulin are substituted with hydrophilic amino acids. In a stillfurther embodiment, two or more hydrophobic amino acids situated twoamino acids away from to two or more protease sites on an insulin aresubstituted with hydrophilic amino acids. In a yet further embodiment,two or more hydrophobic amino acids situated three amino acids away fromtwo or more protease sites on an insulin are substituted withhydrophilic amino acids. In a still further embodiment, two or morehydrophobic amino acids situated up to four amino acids away from two ormore protease sites on an insulin are substituted with hydrophilic aminoacids. In a yet further embodiment two or more hydrophobic amino acidssituated one, two or three amino acids away from or within two or moreprotease sites on an insulin are substituted with hydrophilic aminoacids. In a still further embodiment, two or more hydrophobic aminoacids situated one or two amino acids away from or within two or moreprotease sites on an insulin are substituted with hydrophilic aminoacids. In a yet further embodiment, two or more hydrophobic amino acidssituated next to or within two or more protease sites on an insulin aresubstituted with hydrophilic amino acids.

A protease stabilised insulin may have a net charge which is differentthan the net charge of the parent insulin. In one embodiment, the netcharge of a protease stabilised insulin is more positive than the netcharge of the parent insulin. In one embodiment, the net charge of aprotease stabilised insulin is more negative than the net charge of theparent insulin. In one embodiment, the average positive net charge of aprotease stabilised insulin is between 0.5 and 5 as measured in anaqueous solution. In one embodiment, the average positive net charge ofa protease stabilised insulin is between 1 and 5. In one embodiment, theaverage positive net charge of a protease stabilised insulin is between1 and 4. In one embodiment, the average positive net charge of aprotease stabilised insulin is between 1 and 3. In one embodiment, theaverage positive net charge of a protease stabilised insulin is between2 and 3. In one embodiment, the average negative net charge of aprotease stabilised insulin is between −0.5 and −5 as measured in anaqueous solution. In one embodiment, the average negative net charge ofa protease stabilised insulin is between −1 and −5. In one embodiment,the average negative net charge of a protease stabilised insulin isbetween −1 and −4. In one embodiment, the average negative net charge ofa protease stabilised insulin is between −1 and −3. In one embodiment,the average negative net charge of a protease stabilised insulin isbetween −2 and −3.

In one embodiment, a protease stabilised insulin may have increasedsolubility relative to human insulin. In a further embodiment, aprotease stabilised insulin has increased solubility relative to humaninsulin at pH 3-9. In a yet further embodiment, a protease stabilisedinsulin has increased solubility relative to human insulin at pH 4-8.5.In a still further embodiment, a protease stabilised insulin hasincreased solubility relative to human insulin at pH 4-8. In a yetfurther embodiment, a protease stabilised insulin has increasedsolubility relative to human insulin at pH 4.5-8. In a furtherembodiment, a protease stabilised insulin has increased solubilityrelative to human insulin at pH 5-8. In a yet further embodiment, aprotease stabilised insulin has increased solubility relative to humaninsulin at pH 5.5-8. In a further embodiment, a protease stabilisedinsulin has increased solubility relative to human insulin at pH 6-8.

In one embodiment, a protease stabilised insulin has increasedsolubility relative to human insulin at pH 2-4.

In one embodiment, a protease stabilised insulin may have increasedsolubility relative to the parent insulin. In a further embodiment, aprotease stabilised insulin has increased solubility relative to theparent insulin at pH 3-9. In a yet further embodiment a proteasestabilised insulin has increased solubility relative to parent insulinat pH 4-8.5. In a still further embodiment, a protease stabilisedinsulin has increased solubility relative to parent insulin at pH 4-8.In a yet further embodiment, a protease stabilised insulin has increasedsolubility relative to parent insulin at pH 4.5-8. In a still furtherembodiment, a protease stabilised insulin has increased solubilityrelative to parent insulin at pH 5-8. In a yet further embodiment, aprotease stabilised insulin has increased solubility relative to parentinsulin at pH 5.5-8. In a further embodiment, a protease stabilisedinsulin has increased solubility relative to parent insulin at pH 6-8.

In one embodiment, a protease stabilised insulin has increasedsolubility relative to parent insulin at pH 2-4.

By “increased solubility at a given pH” is meant that a largerconcentration of a protease stabilised insulin dissolves in an aqueousor buffer solution at the pH of the solution relative to the parentinsulin. Methods for determining whether the insulin contained in asolution is dissolved are known in the art.

In one embodiment, the solution may be subjected to centrifugation for20 minutes at 30,000 g and then the insulin concentration in thesupernatant may be determined by RP-HPLC. If this concentration is equalwithin experimental error to the insulin concentration originally usedto make the composition, then the insulin is fully soluble in thecomposition of the invention. In another embodiment, the solubility ofthe insulin in a composition of the invention can simply be determinedby examining by eye the container in which the composition is contained.The insulin is soluble if the solution is clear to the eye and noparticulate matter is either suspended or precipitated on thesides/bottom of the container.

A protease stabilised insulin may have increased apparent potency and/orbioavalability relative to the parent insulin when compared uponmeasurement.

Standard assays for measuring insulin in vitro potency are known to theperson skilled in the art and include inter alia (1) insulinradioreceptorassays, in which the relative potency of an insulin isdefined as the ratio of insulin to insulin analogue required to displace50% of ¹²⁵I-insulin specifically bound to insulin receptors present oncell membranes, e.g., a rat liver plasma membrane fraction; (2)lipogenesis assays, performed, e.g., with rat adipocytes, in whichrelative insulin potency is defined as the ratio of insulin to insulinanalogue required to achieve 50% of the maximum conversion of [3-³H]glucose into organic-extractable material (i.e. lipids); (3) glucoseoxidation assays in isolated fat cells in which the relative potency ofthe insulin analogue is defined as the ratio of insulin to insulinanalogue to achieve 50% of the maximum conversion of glucose-1-[¹⁴C]into [¹⁴CO₂]; (4) insulin radioimmunoassays which can determine theimmunogenicity of insulin analogues by measuring the effectiveness bywhich insulin or an insulin analogue competes with ¹²⁵I-insulin inbinding to specific anti-insulin antibodies; and (5) other assays whichmeasure the binding of insulin or an insulin analogue to antibodies inanimal blood plasma samples, such as ELISA assays possessing specificinsulin antibodies.

Increased apparent in vivo potency can be estimated/visualised bycomparison of blood glucose vs. time profiles of the insulin in questionwith a similar insulin without protease stabilising mutations given insimilar doses. The insulin of the invention will have increased bloodglucose lowering effect relative to the comparator.

Standard assays for measuring insulin bioavailability are known to theperson skilled in the art and include inter alia measurement of therelative areas under the curve (AUC) for the concentration of theinsulin in question administered pulmonary or orally and intra venously(i.v.) in the same species. Quantitation of insulin concentrations inblood (plasma) samples can be done using for example antibody assays(ELISA) or by mass spectrometry. Pulmonary administration can beperformed by several means. For example, insulins can be dosed to ratsby drop instillation, or to pigs by dry powder insufflation.

Protease stabilised insulin may optionally be analyzed for furtherprotease sites which may be subject to further substitutions of one ormore hydrophobic amino acids with hydrophilic amino acids. A proteasestabilised insulin may be an insulin analogue which has at least twohydrophilic acids in protease sites compared to the parent insulin, thefirst modified insulin, and which has further at least one amino acidsubstitution in a new protease site of the first modified insulinwherein at least one hydrophobic amino acid has been substituted with atleast one hydrophilic amino acid.

For the sake of convenience, here follows the names of codable, naturalamino acids with the usual three letter codes & one letter codes inparenthesis: Glycine (Gly & G), proline (Pro & P), alanine (Ala & A),valine (Val & V), leucine (Leu & L), isoleucine (Ile & I), methionine(Met & M), cysteine (Cys & C), phenylalanine (Phe & F), tyrosine (Tyr &Y), tryptophan (Trp & W), histidine (His & H), lysine (Lys & K),arginine (Arg & R), glutamine (Gln & Q), asparagine (Asn & N), glutamicacid (Glu & E), aspartic acid (Asp & D), serine (Ser & S) and threonine(Thr & T). If, due to typing errors, there are deviations from thecommonly used codes, the commonly used codes apply. The amino acidspresent in the insulins of this invention are, preferably, amino acidswhich can be coded for by a nucleic acid. In one embodiment insulin oran insulin analogue is substituted by Gly, Glu, Asp, His, Gln, Asn, Ser,Thr, Lys, Arg and/or Pro and/or Gly, Glu, Asp, His, Gln, Asn, Ser, Thr,Lys, Arg and/or Pro is added to insulin or an insulin analogue. In oneembodiment insulin or an insulin analogue is substituted by Glu, Asp,His, Gln, Asn, Lys and/or Arg and/or Glu, Asp, His, Gln, Asn, Lys and/orArg is added to insulin or an insulin analogue.

In one embodiment, a protease stabilised insulin is selected from thegroup consisting of the following compounds: A14E, B25H, desB30 humaninsulin; A14H, B25H, desB30 human insulin; A14E, B1E, B25H, desB30 humaninsulin; A14E, B16E, B25H, desB30 human insulin; A14E, B25H, B28D,desB30 human insulin; A14E, B25H, B27E, desB30 human insulin; A14E, B1E,B25H, B27E, desB30 human insulin; A14E, B1E, B16E, B25H, B27E, desB30human insulin; A8H, A14E, B25H, desB30 human insulin; A8H, A14E, B25H,B27E, desB30 human insulin; A8H, A14E, B1E, B25H, desB30 human insulin;A8H, A14E, B1E, B25H, B27E, desB30 human insulin; A8H, A14E, B1E, B16E,B25H, B27E, desB30 human insulin; A8H, A14E, B16E, B25H, desB30 humaninsulin; A14E, B25H, B26D, desB30 human insulin; A14E, B1E, B27E, desB30human insulin; A14E, B27E, desB30 human insulin; A14E, B28D, desB30human insulin; A14E, B28E, desB30 human insulin; A14E, B1E, B28E, desB30human insulin; A14E, B1E, B27E, B28E, desB30 human insulin; A14E, B1E,B25H, B28E, desB30 human insulin; A14E, B1E, B25H, B27E, B28E, desB30human insulin; A14D, B25H, desB30 human insulin; B25N, B27E, desB30human insulin; A8H, B25N, B27E, desB30 human insulin; A14E, B27E, B28E,desB30 human insulin; A14E, B25H, B28E, desB30 human insulin; B25H,B27E, desB30 human insulin; B1E, B25H, B27E, desb30 human insulin; A8H,B1E, B25H, B27E, desB30 human insulin; A8H, B25H, B27E, desB30 humaninsulin; B25N, B27D, desB30 human insulin; A8H, B25N, B27D, desB30 humaninsulin; B25H, B27D, desB309 human insulin; A8H, B25H, B27D, desB30human insulin; A(−1)P, A(O)P, A14E, B25H, desB30 human insulin; A14E,B(−1)P, B(O)P, B25H, desB30 human insulin; A(−1)P, A(O)P, A14E, B(−1)P,B(O)P, B25H, desB30 human insulin; A14E, B25H, B30T, B31L, B32E humaninsulin; A14E, B25H human insulin; A14E, B16H, B25H, desB30 humaninsulin; A14E, B10P, B25H, desB30 human insulin; A14E, B10E, B25H,desB30 human insulin; A14E, B4E, B25H, desB30 human insulin; A14H, B16H,B25H, desB30 human insulin; A14H, B10E, B25H, desB30 human insulin;A13H, A14E, B10E, B25H, desB30 human insulin; A13H, A14E, B25H, desB30human insulin; A14E, A18Q, B3Q, B25H, desB30 human insulin; A14E, B24H,B25H, desB30 human insulin; A14E, B25H, B26G, B27G, B28G, desB30 humaninsulin; A14E, B25H, B26G, B27G, B28G, B29R, desB30 human insulin; A14E,A21G, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, A21G, B25H,B26G, B27G, B28G, B29R, desB30 human insulin; A14E, A18Q, A21Q, B3Q,B25H, desB30 human insulin; A14E, A18Q, A21Q, B3Q, B25H, B27E, desB30human insulin; A14E, A18Q, B3Q, B25H, desB30 human insulin; A13H, A14E,B1E, B25H, desB30 human insulin; A13N, A14E, B25H, desB30 human insulin;A13N, A14E, B1E, B25H, desB30 human insulin; A(−2)G, A(−1)P, A(O)P,A14E, B25H, desB30 human insulin; A14E, B(−2)G, B(−1)P, B(O)P, B25H,desB30 human insulin; A(−2)G, A(−1)P, A(O)P, A14E, B(−2)G, B(−1)P,B(O)P, B25H, desB30 human insulin; A14E, B27R, B28D, B29K, desB30 humaninsulin; A14E, B25H, B27R, B28D, B29K, desB30 human insulin; A14E, B25H,B26T, B27R, B28D, B29K, desB30 human insulin; A14E, B25H, B27R, desB30human insulin; A14E, B25H, B27H, desB30 human insulin; A14E, A18Q, B3Q,B25H, desB30 human insulin; A13E, A14E, B25H, desB30 human insulin;A12E, A14E, B25H, desB30 human insulin; A15E, A14E, B25H, desB30 humaninsulin; A13E, B25H, desB30 human insulin; A12E, B25H, desB30 humaninsulin; A15E, B25H, desB30 human insulin; A14E, B25H, desB27, desB30human insulin; A14E, B25H, B26D, B27E, desB30 human insulin; A14E, B25H,B27R, desB30 human insulin; A14E, B25H, B27N, desB30 human insulin;A14E, B25H, B27D, desB30 human insulin; A14E, B25H, B27Q, desB30 humaninsulin; A14E, B25H, B27E, desB30 human insulin; A14E, B25H, B27G,desB30 human insulin; A14E, B25H, B27H, desB30 human insulin; A14E,B25H, B27K, desB30 human insulin; A14E, B25H, B27P, desB30 humaninsulin; A14E, B25H, B27S, desB30 human insulin; A14E, B25H, B27T,desB30 human insulin; A13R, A14E, B25H, desB30 human insulin; A13N,A14E, B25H, desB30 human insulin; A13D, A14E, B25H, desB30 humaninsulin; A13Q, A14E, B25H, desB30 human insulin; A13E, A14E, B25H,desB30 human insulin; A13G, A14E, B25H, desB30 human insulin; A13H,A14E, B25H, desB30 human insulin; A13K, A14E, B25H, desB30 humaninsulin; A13P, A14E, B25H, desB30 human insulin; A13S, A14E, B25H,desB30 human insulin; A13T, A14E, B25H, desB30 human insulin; A14E,B16R, B25H, desB30 human insulin; A14E, B16D, B25H, desB30 humaninsulin; A14E, B16Q, B25H, desB30 human insulin; A14E, B16E, B25H,desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14R,B25H, desB30 human insulin; A14N, B25H, desB30 human insulin; A14D,B25H, desB30 human insulin; A14Q, B25H, desB30 human insulin; A14E,B25H, desB30 human insulin; A14G, B25H, desB30 human insulin; A14H,B25H, desB30 human insulin; A8H, B10D, B25H human insulin; and A8H,A14E, B10E, B25H, desB30 human insulin and this embodiment may,optionally, comprise A14E, B25H, B29R, desB30 human insulin; B25H,desB30 human insulin; and B25N, desB30 human insulin.

In a preferred embodiment, a protease stabilised insulin is selectedfrom the group consisting of the following compounds: A14E, B25H, desB30human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B16E, B25H,desB30 human insulin; A14E, B25H, B29R, desB30 human insulin; A14E,B25H, B26G, B27G, B28G, desB30 human insulin; B25H, desB30 human insulinand A14E, B25H, desB27, desB30 human insulin.

In a preferred embodiment, a protease stabilised insulin is selectedfrom any of the groups above that, in addition, are containing thedesB27 mutation.

In a preferred embodiment, a protease stabilised insulin is selectedfrom the group consisting of the following compounds: A14E, B25H,desB27, desB30 human insulin; A14E, B16H, B25H, desB27, desB30 humaninsulin; A14E, B16E, B25H, desB27, desB30 human insulin; A14E, B25H,desB27, B29R, desB30 human insulin and B25H, desB27, desB30 humaninsulin.

In one embodiment, a protease stabilised insulin is selected from any ofthe groups above that, in addition, are containing the followingmutations in position A21 and/or B3 to improve chemical stability: A21G,desA21, B3Q, or B3G.

In a preferred embodiment, a protease stabilised insulin is selectedfrom the following protease stabilised insulins: A14E, A21G, B25H,desB30 human insulin; A14E, A21G, B16H, B25H, desB30 human insulin;A14E, A21G, B16E, B25H, desB30 human insulin; A14E, A21G, B25H, desB27,desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin;A14E, A21G, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, A21G,B25H, B26G, B27G, B28G, B29R, desB30 human insulin; A21G, B25H, desB30human insulin and A21G, B25N, desB30 human insulin, and, preferably, itis selected from the following protease stabilised insulins: A14E, A21G,B25H, desB30 human insulin; A14E, A21G, B16H, B25H, desB30 humaninsulin; A14E, A21G, B16E, B25H, desB30 human insulin; A14E, A21G, B25H,desB27, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 humaninsulin; A21G, B25H, desB30 human insulin and A21G, B25N, desB30 humaninsulin.

In a preferred embodiment, a protease stabilised insulin is acylated inthe B29 position, at the epsilon nitrogen position of B29K.

In a preferred embodiment, a protease stabilised insulin is acylated inthe A1 position, at the alpha nitrogen position of A1.

In a preferred embodiment, a protease stabilised insulin is acylated inthe A1 position, at the alpha nitrogen position of A1, and the proteasestabilized insulin is comprising the B29R mutation.

The protease stabilised insulins are produced by expressing a DNAsequence encoding the insulin in question in a suitable host cell bywell known technique as disclosed in, e.g., U.S. Pat. No. 6,500,645. Theprotease stabilised insulin is either expressed directly or as aprecursor molecule which has an N-terminal extension on the B-chain.This N-terminal extension may have the function of increasing the yieldof the directly expressed product and may be of up to 15 amino acidresidues long. The N-terminal extension is to be cleaved of in vitroafter isolation from the culture broth and will therefore have acleavage site next to B1. N-terminal extensions of the type suitable inthis invention are disclosed in U.S. Pat. No. 5,395,922, and EuropeanPatent No. 765,395A.

The polynucleotide sequence coding for the protease stabilised insulinmay be prepared synthetically by established standard methods, e.g., thephosphoamidite method described by Beaucage et al. (1981) TetrahedronLetters 22:1859-1869, or the method described by Matthes et al. (1984)EMBO Journal 3: 801-805. According to the phosphoamidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, duplexed and ligated to form the synthetic DNA construct. Acurrently preferred way of preparing the DNA construct is by polymerasechain reaction (PCR).

The polynucleotide sequences may also be of mixed genomic, cDNA, andsynthetic origin. For example, a genomic or cDNA sequence encoding aleader peptide may be joined to a genomic or cDNA sequence encoding theA and B chains, after which the DNA sequence may be modified at a siteby inserting synthetic oligonucleotides encoding the desired amino acidsequence for homologous recombination in accordance with well-knownprocedures or preferably generating the desired sequence by PCR usingsuitable oligonucleotides.

The recombinant method will typically make use of a vector which iscapable of replicating in the selected microorganism or host cell andwhich carries a polynucleotide sequence encoding the protease stabilisedinsulin. The recombinant vector may be an autonomously replicatingvector, i.e., a vector which exists as an extra-chromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, an extra-chromosomal element, a mini-chromosome, or anartificial chromosome. The vector may contain any means for assuringself-replication. Alternatively, the vector may be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. Furthermore, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon may beused. The vector may be linear or closed circular plasmids and willpreferably contain an element(s) that permits stable integration of thevector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

The recombinant expression vector is capable of replicating in yeast.Examples of sequences which enable the vector to replicate in yeast arethe yeast plasmid 2 μm replication genes REP 1-3 and origin ofreplication.

The vector may contain one or more selectable markers which permit easyselection of trans-formed cells. A selectable marker is a gene theproduct of which provides for biocide or viral resistance, resistance toheavy metals, prototrophy to auxotrophs, and the like. Examples ofbacterial selectable markers are the dal genes from Bacillus subtilis orBacillus licheniformis, or markers which confer antibiotic resistancesuch as ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Selectable markers for use in a filamentous fungal host cellinclude amdS (acetamidase), argB (ornithine carbamoyltransferase), pyrG(orotidine-5′-phosphate decarboxylase) and trpC (anthranilate synthase.Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3. A well suited selectable marker for yeast is theSchizosaccharomyces pompe TPI gene (Russell (1985) Gene 40:125-130).

In the vector, the polynucleotide sequence is operably connected to asuitable promoter sequence. The promoter may be any nucleic acidsequence which shows transcriptional activity in the host cell of choiceincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extra-cellular or intra-cellular polypeptides eitherhomologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and Bacilluslicheniformis penicillinase gene (penP). Examples of suitable promotersfor directing the transcription in a filamentous fungal host cell arepromoters obtained from the genes for Aspergillus oryzae TAKA amylase,Rhizomucor miehei aspartic proteinase, Aspergillus niger neutralalpha-amylase, and Aspergillus niger acid stable alpha-amylase. In ayeast host, useful promoters are the Saccharomyces cerevisiae Ma1, TPI,ADH or PGK promoters.

The polynucleotide sequence encoding the protease stabilised insulinwill also typically be operably connected to a suitable terminator. Inyeast a suitable terminator is the TPI terminator (Alber et al. (1982)J. Mol. Appl. Genet. 1:419-434).

The procedures used to ligate the polynucleotide sequence encoding theprotease stabilised insulin, the promoter and the terminator,respectively, and to insert them into a suitable vector containing theinformation necessary for replication in the selected host, are wellknown to persons skilled in the art. It will be understood that thevector may be constructed either by first preparing a DNA constructcontaining the entire DNA sequence encoding the insulins of thisinvention, and subsequently inserting this fragment into a suitableexpression vector, or by sequentially inserting DNA fragments containinggenetic information for the individual elements (such as the signal,pro-peptide, connecting peptide, A and B chains) followed by ligation.

The vector comprising the polynucleotide sequence encoding the proteasestabilised insulin is introduced into a host cell so that the vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication. The host cell may be aunicellular microorganism, e.g., a prokaryote, or a non-unicellularmicroorganism, e.g., a eukaryote. Useful unicellular cells are bacterialcells such as gram positive bacteria including, but not limited to, aBacillus cell, Streptomyces cell, or gram negative bacteria such as E.coli and Pseudomonas sp. Eukaryote cells may be mammalian, insect,plant, or fungal cells. In one embodiment, the host cell is a yeastcell. The yeast organism may be any suitable yeast organism which, oncultivation, produces large amounts of the single chain insulin of theinvention. Examples of suitable yeast organisms are strains selectedfrom the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri,Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis,Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichiakluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candidacacaoi, Geotrichum sp., and Geotrichum fermentans.

The transformation of the yeast cells may for instance be effected byprotoplast formation followed by transformation in a manner known perse. The medium used to cultivate the cells may be any conventionalmedium suitable for growing yeast organisms. The secreted insulin, asignificant proportion of which will be present in the medium incorrectly processed form, may be recovered from the medium byconventional procedures including separating the yeast cells from themedium by centrifugation, filtration or catching the insulin precursorby an ion exchange matrix or by a reverse phase absorption matrix,precipitating the proteinaceous components of the supernatant orfiltrate by means of a salt, e.g., ammonium sulphate, followed bypurification by a variety of chromatographic procedures, e.g., ionexchange chromatography, affinity chromatography, or the like.

Preferably, the acylated insulins of this invention are mono-substitutedhaving only one acylation group attached to a lysine amino acid residuein the protease stabilised insulin molecule.

In one embodiment, the acyl moiety attached to the protease stabilisedinsulin has the general formula:

Acy-AA1_(n)-AA2_(m)-AA3_(p)-  (I),

wherein n is 0 or an integer in the range from 1 to 3; m is 0 or aninteger in the range from 1 to 10; p is 0 or an integer in the rangefrom 1 to 10; Acy is a fatty acid or a fatty diacid comprising fromabout 8 to about 24 carbon atoms; AA1 is a neutral linear or cyclicamino acid residue; AA2 is an acidic amino acid residue; AA3 is aneutral, alkyleneglycol-containing amino acid residue; the order bywhich AA1, AA2 and AA3 appears in the formula can be interchangedindependently; AA2 can occur several times along the formula (e.g.,Acy-AA2-AA3₂-AA2-); AA2 can occur independently (=being different)several times along the formula (e.g., Acy-AA2-AA3₂-AA2-); theconnections between Acy, AA1, AA2 and/or AA3 are amide (peptide) bondswhich, formally, can be obtained by removal of a hydrogen atom or ahydroxyl group (water) from each of Acy, AA1, AA2 and AA3; andattachment to the protease stabilised insulin can be from the C-terminalend of a AA1, AA2, or AA3 residue in the acyl moiety of the formula (I)or from one of the side chain(s) of an AA2 residue present in the moietyof formula (I).

In another embodiment, the acyl moiety attached to the proteasestabilised insulin has the general formulaAcy-AA1_(n)AA2_(m)-AA3_(p)-(I), wherein AA1 is selected from Gly, D- orL-Ala, βAla, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoicacid, D- or L-Glu-α-amide, D- or L-Glu-γ-amide, D- or L-Asp-α-amide, D-or L-Asp-β-amide, or a group of one of the formula:

from which a hydrogen atom and/or a hydroxyl group has been removed andwherein q is 0, 1, 2, 3 or 4 and, in this embodiment, AA1 may,alternatively, be 7-aminoheptanoic acid or 8-aminooctanoic acid.

In another embodiment, the acyl moiety attached to the proteasestabilised insulin has the general formulaAcy-AA1_(n)-AA2_(m)-AA3_(p)-(I), wherein AA1 is as defined above and AA2is selected from L- or D-Glu, L- or D-Asp, L- or D-homoGlu or any of thefollowing:

from which a hydrogen atom and/or a hydroxyl group has been removed andwherein the arrows indicate the attachment point to the amino group ofAA1, AA2, AA3, or to the amino group of the protease stabilised insulin.

In one aspect, the neutral cyclic amino acid residue designated AA1 isan amino acid containing a saturated 6-membered carbocyclic ring,optionally containing a nitrogen hetero atom, and preferably the ring isa cyclohexane ring or a piperidine ring. Preferably, the molecularweight of this neutral cyclic amino acid is in the range from about 100to about 200 Da.

The acidic amino acid residue designated AA2 is an amino acid with amolecular weight of up to about 200 Da comprising two carboxylic acidgroups and one primary or secondary amino group. Alternatively, acidicamino acid residue designated AA2 is an amino acid with a molecularweight of up to about 250 Da comprising one carboxylic acid group andone primary or secondary sulphonamide group.

The neutral, alkyleneglycol-containing amino acid residue designated AA3is an alkyleneglycol moiety, optionally an oligo- or polyalkyleneglycolmoiety containing a carboxylic acid functionality at one end and a aminogroup functionality at the other end.

Herein, the term alkyleneglycol moiety covers mono-alkyleneglycolmoieties as well as oligoalkyleneglycol moieties. Mono- andoligoalkyleneglycols comprises mono- and oligoethyleneglycol based,mono- and oligopropyleneglycol based and mono- and oligobutyleneglycolbased chains, i.e., chains that are based on the repeating unit—CH₂CH₂O—, —CH₂CH₂CH₂O— or —CH₂CH₂CH₂CH₂O—. The alkyleneglycol moiety ismonodisperse (with well defined length/molecular weight).Monoalkyleneglycol moieties comprise —OCH₂CH₂O—, —OCH₂CH₂CH₂O— or—OCH₂CH₂CH₂CH₂O— containing different groups at each end.

As mentioned herein, the order by which AA1, AA2 and AA3 appears in theacyl moiety with the formula (I) (Acy-AA1_(n)-AA2_(m)-AA3_(p)—) can beinterchanged independently. Consequently, the formulaAcy-AA1_(n)-AA2_(m)-AA3_(p)— also covers moieties like, e.g., theformula Acy-AA2_(m)-AA1-AA3_(p)—, the formula Acy-AA2-AA3_(n)-AA2-, andthe formula Acy-AA3_(p)-AA2_(m)-AA1_(n)-, wherein Acy, AA1, AA2, AA3, n,m and p are as defined herein.

As mentioned herein, the connections between the moieties Acy, AA1, AA2and/or AA3 are formally obtained by amide bond (peptide bond) formation(—CONH—) by removal of water from the parent compounds from which theyformally are build. This means that in order to get the complete formulafor the acyl moiety with the formula (I) (Acy-AA1_(n)-AA2_(m)-AA3_(p)—,wherein Acy, AA1, AA2, AA3, n, m and p are as defined herein), one has,formally, to take the compounds given for the terms Acy, AA1, AA2 andAA3 and remove a hydrogen and/or hydroxyl from them and, formally, toconnect the building blocks so obtained at the free ends so obtained.

Non-limiting, specific examples of the acyl moieties of the formulaAcy-AA1_(n)-AA2_(m)-AA3_(p)-which may be present in the acylated insulinanalogues of this invention are the following:

Any of the above non-limiting specific examples of acyl moieties of theformula Acy-AA1_(n)-AA2_(m)-AA3_(p)- can be attached to an epsilon aminogroup of a lysine residue present in any of the above non-limitingspecific examples of insulin analogues thereby giving further specificexamples of acylated insulin analogues of this invention.

Any of the above non-limiting specific examples of acyl moieties of theformula Acy-AA1_(n)-AA2_(m)-AA3_(p)- can be attached to an alpha aminogroup of a A1 residue present in any of the above non-limiting specificexamples of insulin analogues thereby giving further specific examplesof acylated insulin analogues of this invention.

The protease stabilized insulins can be converted into the acylatedprotease stabilized insulins of this invention by introducing of thedesired group of the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)- in the lysineresidue or in a N-terminal position in the insulin analogue. The desiredgroup of the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)- can be introduced byany convenient method and many methods are disclosed in the prior artfor such reactions. More details appear from the examples herein.

In an embodiment, the present invention does not relate to compoundsdescribed in EP 07114387.9, i.e., acylated insulins wherein an acylmoiety is attached to the parent insulin and wherein said acyl moietycomprises repeating units of alkylene glycol containing amino acids andwherein there is only one lysine residue (K & Lys) in the parentinsulin.

Pharmaceutical Compositions

The acylated insulins of this invention may be administeredsubcutaneously, nasally, orally, or pulmonary.

For subcutaneous administration, the acylated insulins of this inventionare formulated analogously with the formulation of known insulins.Furthermore, for subcutaneous administration, the acylated insulins ofthis invention are administered analogously with the administration ofknown insulins and, generally, the physicians are familiar with thisprocedure.

Acylated insulins of this invention may be administered by inhalation ina dose effective to increase circulating insulin levels and/or to lowercirculating glucose levels. Such administration can be effective fortreating disorders such as diabetes or hyperglycemia. Achievingeffective doses of insulin requires administration of an inhaled dose ofmore than about 0.5 μg/kg to about 50 μg/kg of acylated insulins of thisinvention. A therapeutically effective amount can be determined by aknowledgeable practitioner, who will take into account factors includinginsulin level, blood glucose levels, the physical condition of thepatient, the patient's pulmonary status, or the like.

The acylated insulins of this invention may be delivered by inhalationto achieve slow absorption and/or reduced systemical clearance thereof.Different inhalation devices typically provide similar pharmacokineticswhen similar particle sizes and similar levels of lung deposition arecompared.

The acylated insulins of this invention may be delivered by any of avariety of inhalation devices known in the art for administration of atherapeutic agent by inhalation. These devices include metered doseinhalers, nebulizers, dry powder generators, sprayers, and the like.Preferably, the acylated insulins of this are delivered by a dry powderinhaler or a sprayer. There are a several desirable features of aninhalation device for administering acylated insulins of this invention.For example, delivery by the inhalation device is advantageouslyreliable, reproducible, and accurate. The inhalation device shoulddeliver small particles or aerosols, e.g., less than about 10 μm, forexample about 1-5 μm, for good respirability. Some specific examples ofcommercially available inhalation devices suitable for the practice ofthis invention are Turbohaler™ (Astra), Rotahaler® (Glaxo), Diskus®(Glaxo), Spiros™ inhaler (Dura), devices marketed by InhaleTherapeutics, AERx™ (Aradigm), the Ultravent® nebulizer (Mallinckrodt),the Acorn II® nebulizer (Marquest Medical Products), the Ventolin®metered dose inhaler (Glaxo), the Spinhaler® powder inhaler (Fisons), orthe like.

As those skilled in the art will recognize, the formulation of acylatedinsulins of this invention, the quantity of the formulation deliveredand the duration of administration of a single dose depend on the typeof inhalation device employed. For some aerosol delivery systems, suchas nebulizers, the frequency of administration and length of time forwhich the system is activated will depend mainly on the concentration ofacylated insulins in the aerosol. For example, shorter periods ofadministration can be used at higher concentrations of acylated insulinsin the nebulizer solution. Devices such as metered dose inhalers canproduce higher aerosol concentrations, and can be operated for shorterperiods to deliver the desired amount of the acylated insulins. Devicessuch as powder inhalers deliver active agent until a given charge ofagent is expelled from the device. In this type of inhaler, the amountof insulin acylated insulins of this invention in a given quantity ofthe powder determines the dose delivered in a single administration.

The particle size of acylated insulins of this invention in theformulation delivered by the inhalation device is critical with respectto the ability of insulin to make it into the lungs, and preferably intothe lower airways or alveoli. Preferably, the acylated insulins of thisinvention ion is formulated so that at least about 10% of the acylatedinsulins delivered is deposited in the lung, preferably about 10 toabout 20%, or more. It is known that the maximum efficiency of pulmonarydeposition for mouth breathing humans is obtained with particle sizes ofabout 2 μm to about 3 μm. When particle sizes are above about 5 μm,pulmonary deposition decreases substantially. Particle sizes below about1 μm cause pulmonary deposition to decrease, and it becomes difficult todeliver particles with sufficient mass to be therapeutically effective.Thus, particles of the acylated insulins delivered by inhalation have aparticle size preferably less than about 10 μm, more preferably in therange of about 1 μm to about 5 μm. The formulation of the acylatedinsulins is selected to yield the desired particle size in the choseninhalation device.

Advantageously for administration as a dry powder an acylated insulin ofthis invention is prepared in a particulate form with a particle size ofless than about 10 μm, preferably about 1 to about 5 μm. The preferredparticle size is effective for delivery to the alveoli of the patient'slung. Preferably, the dry powder is largely composed of particlesproduced so that a majority of the particles have a size in the desiredrange. Advantageously, at least about 50% of the dry powder is made ofparticles having a diameter less than about 10 μm. Such formulations canbe achieved by spray drying, milling, or critical point condensation ofa solution containing the acylated insulin of this invention and otherdesired ingredients. Other methods also suitable for generatingparticles useful in the current invention are known in the art.

The particles are usually separated from a dry powder formulation in acontainer and then transported into the lung of a patient via a carrierair stream. Typically, in current dry powder inhalers, the force forbreaking up the solid is provided solely by the patient's inhalation. Inanother type of inhaler, air flow generated by the patient's inhalationactivates an impeller motor which deagglomerates the particles.

Formulations of acylated insulins of this invention for administrationfrom a dry powder inhaler typically include a finely divided dry powdercontaining the derivative, but the powder can also include a bulkingagent, carrier, excipient, another additive, or the like. Additives canbe included in a dry powder formulation of acylated insulin, e.g., todilute the powder as required for delivery from the particular powderinhaler, to facilitate processing of the formulation, to provideadvantageous powder properties to the formulation, to facilitatedispersion of the powder from the inhalation device, to stabilize theformutation (for example, antioxidants or buffers), to provide taste tothe formulation, or the like. Advantageously, the additive does notadversely affect the patient's airways. The acylated insulin can bemixed with an additive at a molecular level or the solid formulation caninclude particles of the acylated insulin mixed with or coated onparticles of the additive. Typical additives include mono-, di-, andpolysaccharides; sugar alcohols and other polyols, such as, e.g.,lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose,sucrose, mannitol, starch, or combinations thereof; surfactants, such assorbitols, diphosphatidyl choline, or lecithin; or the like. Typicallyan additive, such as a bulking agent, is present in an amount effectivefor a purpose described above, often at about 50% to about 90% by weightof the formulation. Additional agents known in the art for formulationof a protein such as insulin analogue protein can also be included inthe formulation.

A spray including the acylated insulins of this invention can beproduced by forcing a suspension or solution of the acylated insulinthrough a nozzle under pressure. The nozzle size and configuration, theapplied pressure, and the liquid feed rate can be chosen to achieve thedesired output and particle size. An electrospray can be produced, e.g.,by an electric field in connection with a capillary or nozzle feed.Advantageously, particles of insulin conjugate delivered by a sprayerhave a particle size less than about 10 μm, preferably in the range ofabout 1 μm to about 5 μm.

Formulations of acylated insulins of this invention suitable for usewith a sprayer will typically include the acylated insulins in anaqueous solution at a concentration of from about 1 mg to about 500 mgof the acylated insulin per ml of solution. Depending on the acylatedinsulin chosen and other factors known to the medical advisor, the upperlimit may be lower, e.g., 450, 400, 350, 300, 250, 200, 150, 120, 100 or50 mg of the acylated insulin per ml of solution. The formulation caninclude agents such as an excipient, a buffer, an isotonicity agent, apreservative, a surfactant, and, preferably, zinc. The formulation canalso include an excipient or agent for stabilization of the acylatedinsulin, such as a buffer, a reducing agent, a bulk protein, or acarbohydrate. Bulk proteins useful in formulating insulin conjugatesinclude albumin, protamine, or the like. Typical carbohydrates useful informulating the acylated insulin include sucrose, mannitol, lactose,trehalose, glucose, or the like. The acylated insulins formulation canalso include a surfactant, which can reduce or prevent surface-inducedaggregation of the insulin conjugate caused by atomization of thesolution in forming an aerosol. Various conventional surfactants can beemployed, such as polyoxyethylene fatty acid esters and alcohols, andpolyoxyethylene sorbitol fatty acid esters. Amounts will generally rangebetween about 0.001 and about 4% by weight of the formulation.

Pharmaceutical compositions containing an acylated insulin of thisinvention may also be administered parenterally to patients in need ofsuch a treatment. Parenteral administration may be performed bysubcutaneous, intramuscular or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump.

Injectable compositions of the acylated insulins of this invention canbe prepared using the conventional techniques of the pharmaceuticalindustry which involve dissolving and mixing the ingredients asappropriate to give the desired end product. Thus, according to oneprocedure, an acylated insulin is dissolved in an amount of water whichis somewhat less than the final volume of the composition to beprepared. Zink, an isotonic agent, a preservative and/or a buffer is/areadded as required and the pH value of the solution is adjusted—ifnecessary—using an acid, e.g., hydrochloric acid, or a base, e.g.,aqueous sodium hydroxide as needed. Finally, the volume of the solutionis adjusted with water to give the desired concentration of theingredients.

In a further embodiment of this invention the buffer is selected fromthe group consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of this invention.

In a further embodiment of this invention the formulation furthercomprises a pharmaceutically acceptable preservative which may beselected from the group consisting of phenol, o-cresol, m-cresol,p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzylalcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid,imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethylp-hydroxybenzoate, benzethonium chloride, chlorphenesine(3-(4-chlorophenoxy)-1,2-propanediol) or mixtures thereof. In a furtherembodiment of this invention the preservative is present in aconcentration from about 0.1 mg/ml to 20 mg/ml. In a further embodimentof this invention the preservative is present in a concentration fromabout 0.1 mg/ml to 5 mg/ml. In a further embodiment of this inventionthe preservative is present in a concentration from about 5 mg/ml to 10mg/ml. In a further embodiment of this invention the preservative ispresent in a concentration from about 10 mg/ml to 20 mg/ml. Each one ofthese specific preservatives constitutes an alternative embodiment ofthis invention. The use of a preservative in pharmaceutical compositionsis well-known to the skilled person. For convenience reference is madeto Remington: The Science and Practice of Pharmacy, 19^(th) edition,1995.

In a further embodiment of this invention, the formulation furthercomprises an isotonic agent which may be selected from the groupconsisting of a salt (e.g., sodium chloride), a sugar or sugar alcohol,an amino acid (for example, L-glycine, L-histidine, arginine, lysine,isoleucine, aspartic acid, tryptophan or threonine), an alditol (e.g.glycerol (glycerine), 1,2-propanediol (propyleneglycol), 1,3-propanediolor 1,3-butanediol), polyethyleneglycol (e.g., PEG400) or mixturesthereof. Any sugar such as mono-, di-, or polysaccharides, orwater-soluble glucans, including for example fructose, glucose, mannose,sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran,pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch andcarboxymethylcellulose-Na may be used. In one embodiment the sugaradditive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbonhaving at least one —OH group and includes, e.g., mannitol, sorbitol,inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodimentthe sugar alcohol additive is mannitol. The sugars or sugar alcoholsmentioned above may be used individually or in combination. There is nofixed limit to the amount used, as long as the sugar or sugar alcohol issoluble in the liquid preparation and does not adversely effect thestabilizing effects achieved using the methods of this invention. In oneembodiment, the sugar or sugar alcohol concentration is between about 1mg/ml and about 150 mg/ml. In a further embodiment of this invention theisotonic agent is present in a concentration from about 1 mg/ml to 50mg/ml. In a further embodiment of this invention the isotonic agent ispresent in a concentration from about 1 mg/ml to 7 mg/ml. In a furtherembodiment of this invention the isotonic agent is present in aconcentration from about 8 mg/ml to 24 mg/ml. In a further embodiment ofthis invention the isotonic agent is present in a concentration fromabout 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agentsconstitutes an alternative embodiment of this invention. The use of anisotonic agent in pharmaceutical compositions is well-known to theskilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995. Typicalisotonic agents are sodium chloride, mannitol, dimethyl sulfone andglycerol and typical preservatives are phenol, m-cresol, methylp-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are sodium acetate, glycylglycine, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodiumphosphate.

A composition for nasal administration of an acylated insulins of thisinvention may, e.g., be prepared as described in European Patent No.272,097.

Oral preparations containing an acylated protease stabilised insulin ofthis inventions can be prepared in a manner known per se. One way ofmaking preparations containing an acylated protease stabilised insulinof this invention which can conveniently be administered orally is byusing a procedure which is analagous to the process described in WO2008/145728.

Another way of preparing oral preparations containing an acylatedprotease stabilised insulin of this invention is to prepare a water-freeliquid or semisolid pharmaceutical compositions comprising an acylatedprotease stabilised insulin of this invention (a), at least one polarorganic solvent (b) for the acylated protease stabilised insulin, atleast one lipophilic component (c), and optionally a surfactant (d)and/or at least one solid hydrophilic component (e). This could be inthe form of an oily solution. Alternatively, the at least one solidhydrophilic component (d) is at least one solid hydrophilic polymer.Alternatively, the pharmaceutical composition comprising at least onesolid hydrophilic component is free of surfactant, wherein saidsurfactant has an HLB value which is at least 8, i.e. there is nosurfactant, which has an HLB value which is at least 8, present in thecomposition.

For example, a pharmaceutical composition containing an acylatedprotease stabilised insulin may be a water-free oily solution and/or aSEDDS or SMEDDS pharmaceutical composition.

Alternatively said pharmaceutical composition is a self emulsifying drugdelivery system (herein designated SEDDS).

It is believed that the high solubility of an acylated proteasestabilised insulin in the polar organic solvent of the pharmaceuticalcomposition resulting in the relatively low total amount of polarorganic solvent needed in said pharmaceutical composition may improvecompatibility of the pharmaceutical composition with capsule materials.

The pharmaceutical composition may contain a carrier that comprises alipophilic component, a surfactant and a polar organic solvent andoptionally a solid hydrophilic component (e). If there is a solidhydrophilic component present, at least one of the components selectedfrom the group consisting of a lipophilic component and a surfactant isliquid or semi-solid. If there is a liquid hydrophilic component (e)present, both the lipophilic component and the surfactant may be solid.For example, the surfactant is liquid or semisolid. In one aspect, asolid hydrophilic component is present.

As used herein, the term “carrier” refers to the pharmaceuticallyacceptable vehicle that transports the therapeutically activewater-soluble polypeptide across the biological membrane or within abiological fluid. The carrier comprises a lipophilic component and apolar organic solvent, and optionally a solid hydrophilic componentand/or a surfactant. The carrier is capable of spontaneously producingan emulsion or colloidal structures, when brought in contact, dispersed,or diluted, with an aqueous medium, e.g., water, fluids containingwater, or in vivo media in mammals, such as the gastric juices of thegastrointestinal tract. The colloidal structures can be solid or liquidparticles including domains, droplets, micelles, mixed micelles,vesicles and nanoparticles.

For example, when the pharmaceutical composition is brought into contactwith an aqueous medium, an emulsion, such as a microemulsion,spontaneously forms. In particular, an emulsion or microemulsion formsin the digestive tract of a mammal when the delivery system is orallyingested. In addition to the aforementioned components, thespontaneously dispersible preconcentrate can also optionally containother excipients, such as buffers, pH adjusters, stabilizers and otheradjuvants recognized by one of ordinary skill in the art to beappropriate for such a pharmaceutical use.

The term “water-free” as used herein refers to a composition to which nowater is added during preparation of the pharmaceutical composition. Theacylated protease stabilised insulin and/or one or more of theexcipients in the pharmaceutical composition may have small amounts ofwater bound to it before preparing a pharmaceutical composition. Foreexample, a water-free pharmaceutical composition comprises less than 10%w/w water, for example, less than 5% w/w water, for example, less than4% w/w water, for example, less than 3% w/w water, for example, lessthan 2% w/w water, for example, less than 1% w/w water.

As used herein, the term “microemulsion preconcentrate” means acomposition, which spontaneously forms a microemulsion, e.g., anoil-in-water microemulsion, in an aqueous medium, e.g., in water or inthe gastrointestinal fluids after oral application. The compositionself-emulsifies upon dilution in an aqueous medium for example in adilution of 1:5, 1:10, 1:50, 1:100 or higher.

Due to the high solubility of the acylated protease stabilised insulin,the total amount of polar organic solvent in the SEDDS can be kept lowwhich on the one hand improves compatibility of the formulation withcapsule materials and on the other hand gives more design space for thecomposition.

The pharmaceutical composition comprises a lipophilic component, and anorganic polar component. The components of the drug delivery system canbe present in any relative amounts. For example, the drug deliverysystem can comprises up to 40% polar organic component by weight of thecomposition of the carrier, e.g., less than 30%, 20%, 15% or 10%. Inanother aspect, the drug delivery system comprises from 5% to 40% byweight polar organic solvent of the total composition of the carrier. Inyet a further aspect, the drug delivery system comprises from 10% to 30%by weight polar organic solvent of the total composition of the carrier.

The pharmaceutical composition may be in the form of a non-powdercomposition, i.e. in a semi-solid or liquid form.

As used herein, the term “liquid” means a component or composition thatis in a liquid state at room temperature (“RT”), and having a meltingpoint of, for example, below 20° C. As used herein room temperature (RT)means approximately 20-25° C.

As used herein, the term “semi-solid” relates to a component orcomposition which is not liquid at room temperature, e.g., having amelting point between room temperature and about 40° C. A semisolid canhave the qualities and/or attributes of both the solid and liquid statesof matter. As used-herein, the term “solidify” means to make solid orsemi-solid.

Examples of semi-solid or liquid compositions are pharmaceuticalcompositions in the form of, e.g., oils, solutions, liquid or semisolidSMEDDS and liquid or semisolid SEDDS.

“SMEDDS” (being an abbreviation for self-micro-emulsifying drug deliverysystems) are herein defined as isotropic mixtures of a hydrophiliccomponent, a surfactant, optionally a cosurfactant and a drug thatrapidly form an oil in water microemulsion when exposed to aqueous mediaunder conditions of gentle agitation or digestive motility that would beencountered in the GI tract.

“SEDDS” (being an abbreviation for self emulsifying drug deliverysystems) are herein defined as mixtures of a hydrophilic component, asurfactant, optionally a cosurfactant and a drug that formsspontaneously a fine oil in water emulsion when exposed to aqueous mediaunder conditions of gentle agitation or digestive motility that would beencountered in the GI tract.

As used herein, the term “microemulsion” refers to a clear ortranslucent, slightly opaque, opalescent, non-opaque or substantiallynon-opaque colloidal dispersion that is formed spontaneously orsubstantially spontaneously when its components are brought into contactwith an aqueous medium.

As used herein, the term “emulsion” refers to a slightly opaque,opalescent or opague colloidal dispersion that is formed spontaneouslyor substantially spontaneously when its components are brought intocontact with an aqueous medium.

A microemulsion is thermodynamically stable and contains homogenouslydispersed particles or domains, for example of a solid or liquid state(e.g., liquid lipid particles or droplets), of a mean diameter of lessthan about 500 nm, e.g., less than about 400 nm or less than 300 nm,less than 200 nm, less than 100 nm, and greater than about 2-4 nm asmeasured by standard light scattering techniques, e.g., using a MALVERNZETASIZER Nano ZS. The term “domain size” as used herein refers torepetitive scattering units and can be measured by, e.g., small angleX-ray. In one aspect, the domain size is smaller than 400 nm, in anotheraspect, smaller than 300 nm and in yet another aspect, smaller than 200nm.

As used herein the term “spontaneously dispersible” when referring to apre-concentrate refers to a composition that is capable of producingcolloidal structures such as microemulsions, emulsions and othercolloidal systems, when diluted with an aqueous medium when thecomponents of the composition are brought into contact with an aqueousmedium, e.g., by simple shaking by hand for a short period of time, forexample for ten seconds. In one aspect a spontaneously dispersibleconcentrate according to the invention is a SEDDS or SMEDDS.

As used herein, the term “lipophilic component” refers to a substance,material or ingredient that is more compatible with oil than with water.A material with lipophilic properties is insoluble or almost insolublein water but is easily soluble in oil or other nonpolar solvents. Theterm “lipophilic component” can comprise one or more lipophilicsubstances. Multiple lipophilic components may constitute the lipophilicphase of the spontaneously dispersible preconcentrate and form the oilaspect, e.g., in an oil-in-water emulsion or microemulsion. At roomtemperature, the lipophilic component and lipophilic phase of thespontaneously dispersible preconcentrate can be solid, semisolid orliquid. For example, a solid lipophilic component can exist as a paste,granular form, powder or flake. If more than one excipient comprises thelipophilic component, the lipophilic component can be a mixture ofliquids, solids, or both.

In one aspect, the lipophilic component is present in the pharmaceuticalcomposition in an amount of at least 20% w/w. In a further aspect, thelipophilic component is present in an amount of at least 30%, at least50%, at least 80% or at least 90% w/w. For example, the lipophiliccomponent may be present from about 5% to about 90% by weight of thecomposition, e.g., from about 15% to about 60%, e.g., from about 20% toabout 40%. Examples of solid lipophilic components, i.e., lipophiliccomponents which are solid or semisolid at room temperature, include,but are not limited to, the following:

1. mixtures of mono-, di- and triglycerides, such as hydrogenatedcoco-glycerides (melting point (m.p.) of about 33.5° C. to about 37°C.], commercially-available as WITEPSOL HIS from Sasol Germany (Witten,Germany); Examples of fatty acid triglycerides e.g., C10-C22 fatty acidtriglycerides include natural and hydrogenated oils, such as vegetableoils;

2. esters, such as propylene glycol (PG) stearate, commerciallyavailable as MONOSTEOL (m.p. of about 33° C. to about 36° C.) fromGattefosse Corp. (Paramus, N.J.); diethylene glycol palmito stearate,commercially available as HYDRINE (m.p. of about 44.5° C. to about 48.5°C.) from Gattefosse Corp.;

3. polyglycosylated saturated glycerides, such as hydrogenated palm/palmkernel oil PEG-6 esters (m.p. of about 30.5° C. to about 38° C.),commercially-available as LABRAFIL M2130 CS from Gattefosse Corp. orGelucire 33/01;

4. fatty alcohols, such as myristyl alcohol (m.p. of about 39° C.),commercially available as LANETTE 14 from Cognis Corp. (Cincinnati,Ohio); esters of fatty acids with fatty alcohols, e.g., cetyl palmitate(m.p. of about 50° C.); isosorbid monolaurate, e.g., commerciallyavailable under the trade name ARLAMOL ISML from Uniqema (New Castle,Del.), e.g. having a melting point of about 43° C.;

5. PEG-fatty alcohol ether, including polyoxyethylene (2) cetyl ether,e.g. commercially available as BRIJ 52 from Uniqema, having a meltingpoint of about 33° C., or polyoxyethylene (2) stearyl ether, e.g.commercially available as BRIJ 72 from Uniqema having a melting point ofabout 43° C.;

6. sorbitan esters, e.g. sorbitan fatty acid esters, e.g. sorbitanmonopalmitate or sorbitan monostearate, e.g, commercially available asSPAN 40 or SPAN 60 from Uniqema and having melting points of about 43°C. to 48° C. or about 53° C. to 57° C. and 41° C. to 54° C.,respectively; and

7. glyceryl mono-C6-C14-fatty acid esters. These are obtained byesterifying glycerol with vegetable oil followed by moleculardistillation. Monoglycerides include, but are not limited to, bothsymmetric (i.e. β-monoglycerides) as well as asymmetric monoglycerides(α-monoglycerides). They also include both uniform glycerides (in whichthe fatty acid constituent is composed primarily of a single fatty acid)as well as mixed glycerides (i.e. in which the fatty acid constituent iscomposed of various fatty acids). The fatty acid constituent may includeboth saturated and unsaturated fatty acids having a chain length of frome.g. C8-C14. Particularly suitable are glyceryl mono laurate e.g.commercially available as IMWITOR 312 from Sasol North America (Houston,Tex.), (m.p. of about 56° C.-60° C.); glyceryl mono dicocoate,commercially available as IMWITOR 928 from Sasol (m.p. of about 33°C.-37° C.); monoglyceryl citrate, commercially available as IMWITOR 370,(m.p. of about 59 to about 63° C.); or glyceryl mono stearate, e.g.,commercially available as IMWITOR 900 from Sasol (rn.p. of about 56°C.-61° C.); or self-emulsifying glycerol mono stearate, e.g.,commercially available as IMWITOR 960 from Sasol (m.p. of about 56°C.-61° C.).

Examples of liquid lipophilic components, i.e., lipophilic componentswhich are liquid at room temperature include, but are not limited to,the following:

1. mixtures of mono-, di- and triglycerides, such as medium chain mono-and diglycerides, glyceryl caprylate/caprate, commercially-available asCAPMUL MCM from Abitec Corp. (Columbus, Ohio);

2. glyceryl mono- or di fatty acid ester, e.g. of C6-C18, e.g. C6-C16e.g. C8-C10, e.g. C8, fatty acids, or acetylated derivatives thereof,e.g. MYVACET 9-45 or 9-08 from Eastman Chemicals (Kingsport, Tenn.) orIMWITOR 308 or 312 from Sasol;

3. propylene glycol mono- or di-fatty acid ester, e.g. of C8-C20, e.g.C8-C12, fatty acids, e.g. LAUROGLYCOL 90, SEFSOL 218, or CAPRYOL 90 orCAPMUL PG-8 (same as propylene glycol caprylate) from Abitec Corp.;

4. oils, such as safflower oil, sesame oil, almond oil, peanut oil, palmoil, wheat germ oil, corn oil, castor oil, coconut oil, cotton seed oil,soybean oil, olive oil and mineral oil;

5. fatty acids or alcohols, e.g. C8-C20, saturated or mono- ordi-unsaturated, e.g. oleic acid, oleyl alcohol, linoleic acid, capricacid, caprylic acid, caproic acid, tetradecanol, dodecanol, decanol;

6. medium chain fatty acid triglycerides, e.g. C8-C12, e.g. MIGLYOL 812,or long chain fatty acid triglycerides, e.g. vegetable oils;

7. transesterified ethoxylated vegetable oils, e.g. commerciallyavailable as LABRAFIL M2125 CS from Gattefosse Corp;

8. esterified compounds of fatty acid and primary alcohol, e.g. C8-C20,fatty acids and C2-C3 alcohols, e.g. ethyl linoleate, e.g. commerciallyavailable as NIKKOL VF-E from Nikko Chemicals (Tokyo, Japan), ethylbutyrate, ethyl caprylate oleic acid, ethyl oleate, isopropyl myristateand ethyl caprylate;

9. essential oils, or any of a class of volatile oils that give plantstheir characteristic odors, such as spearmint oil, clove oil, lemon oiland peppermint oil;

10. fractions or constituents of essential oils, such as menthol,carvacrol and thymol;

11. synthetic oils, such as triacetin, tributyrin;

12. triethyl citrate, acetyl triethyl citrate, tributyl citrate, acetyltributyl citrate;

13. polyglycerol fatty acid esters, e.g. diglyceryl monooleate, e.g.DGMO-C, DGMO-90, DGDO from Nikko Chemicals; and

14. sorbitan esters, e.g. sorbitan fatty acid esters, e.g. sorbitanmonolaurate, e.g. commercially available as SPAN 20 from Uniqema.

15. Phospholipids, e.g. Alkyl-O-Phospholipids, Diacyl PhosphatidicAcids, Diacyl Phosphatidyl Cholines, Diacyl Phosphatidyl Ethanolamines,Diacyl Phosphatidyl Glycerols, Di-O-Alkyl Phosphatidic Acids,L-alpha-Lysophosphatidylcholines (LPC),L-alpha-Lysophosphatidylethanolamines (LPE),L-alpha-Lysophosphatidylglycerol (LPG),L-alpha-Lysophosphatidylinositols (LPI), L-alpha-Phosphatidic acids(PA), L-alpha-Phosphatidylcholines (PC),L-alpha-Phosphatidylethanolamines (PE), L-alpha-Phosphatidylglycerols(PG), Cardiolipin (CL), L-alpha-Phosphatidylinositols (PI),L-alpha-Phosphatidylserines (PS), Lyso-Phosphatidylcholines,Lyso-Inventor: Phosphatidylglycerols, sn-Glycerophosphorylcholinescommercially available from LARODAN, or soybean phospholipid (LipoidS100) commercially available from Lipoid GmbH.

For example, the lipophilic component is one or more selected from thegroup consisting of mono-, di-, and triglycerides. In one aspect, thelipophilic component is one or more selected from the group consistingof mono- and diglycerides. In yet a further aspect, the lipophiliccomponent is Capmul MCM or Capmul PG-8. In a still further aspect, thelipophilic component is Capmul PG-8.

The term “polar organic solvent” refers in one aspect herein to a “polarprotic organic solvent” which is a hydrophilic, water misciblecarbon-containing solvent that contains an O—H or N—H bond, or mixturesthereof. The polarity is reflected in the dielectric constant or thedipole moment of a solvent. The polarity of a solvent determines whattype of compounds it is able to dissolve and with what other solvents orliquid compounds it is miscible. Typically, polar organic solventsdissolve polar compounds best and non-polar solvents dissolve non-polarcompounds best: “like dissolves like”. Strongly polar compounds likeinorganic salts (e.g. sodium chloride) dissolve only in very polarsolvents.

Polar organic solvents may be selected from solvent wherein the acylatedproteases stabilised insulin show better solubility in said polarorganic solvents than in other solvents.

Hence, the acylated proteases stabilised insulin can be dissolved to ahigh degree in a water-free pharmaceutical acceptable polar organicsolvent such as propylene glycol, glycerol and PEG200. For example, atleast 20% (w/w) of the acylated proteases stabilised insulin dissolve ina water-free pharmaceutical acceptable polar organic solvent, i.e. whenadding 20% w/w of the acylated proteases stabilised insulin to the polarorganic solvent, a clear solution is obtained. In another aspect, atleast 25%, 30%, 40% or 50% (w/w) of the acylated proteases stabilisedinsulin dissolve in a water-free pharmaceutical acceptable polar organicsolvent.

The polar organic solvent may thus refer to a hydrophilic, watermiscible carbon-containing solvent that contains an O—H or N—H bond, ormixtures thereof. The polarity is reflected in the dielectric constantor the dipole moment of a solvent. The polarity of a solvent determineswhat type of compounds it is able to dissolve and with what othersolvents or liquid compounds it is miscible. Typically, polar solventsdissolve polar compounds best and non-polar solvents dissolve non-polarcompounds best: “like dissolves like”. Strongly polar compounds likeinorganic salts (e.g. sodium chloride) dissolve only in very polarsolvents.

For example, the polar organic solvent is a solvent having a dielectricconstant above 20, preferably in the range of 20-50. Examples ofdifferent polar organic solvent are listed in Table 1 together withwater as a reference.

TABLE 1 Dielectric constants (static permittivity) of selected polarorganic solvents and water as a reference (Handbook of Chemistry andPhysics, CMC Press, dielectric constants are measured in static electricfields or at relatively low frequencies, where no relaxation occurs)Solvent (Temperature, Kelvin) Dielectric constant, ∈* Water (293.2) 80.1Propanetriol [Glycerol] (293.2) 46.53 Ethanediol [Ethylene Glycol](293.2) 41.4 1,3-propanediol (293.2) 35.1 Methanol (293.2) 33.01,4-butanediol (293.2) 31.9 1,3-butanediol (293.2) 28.8 1,2-propanediol[propylene glycol] (303.2) 27.5 Ethanol (293.2) 25.3 Isopropanol[2-propanol, isopropyl alcohol] 20.18 (293.2)

In the present context, 1,2-propanediol and propylene glycol is usedinterchangeably. In the present context, propanetriol and glycerol isused interchangeably. In the present context, ethanediol and ethyleneglycol is used interchangeably.

For example, the polar organic solvent is selected from the groupconsisting of polyols. The term “polyol” as used herein refers tochemical compounds containing multiple hydroxyl groups.

In one aspect, the polar organic solvent is selected from the groupconsisting of diols and triols. The term “diol” as used herein refers tochemical compounds containing two hydroxyl groups. The term “triol” asused herein refers to chemical compounds containing three hydroxylgroups.

For example, the polar organic solvent is selected from the groupconsisting of glycerol (propanetriol), ethanediol (ethylene glycol),1,3-propanediol, methanol, 1,4-butanediol, 1,3-butanediol, propyleneglycol (1,2-propanediol), ethanol and isopropanol, or mixtures thereof.In one alternative, the polar organic solvent is selected from the groupconsisting of propylene glycol and glycerol. Glycerol is biocompatibleeven at high dosages and has a high solvent capacity for the acylatedproteasde stabilised insulin. Alternatively, the polar organic solventis selected from the group consisting of propylene glycol and ethyleneglycol. These polar organic solvent have a low viscosity, arebiocompatible at moderate doses, and have very high polar organicsolvent capacity for the acylated proteasde stabilised insulin.

The polar organic solvent should preferably be of high purity with a lowcontent of, e.g., aldehydes, ketones and other reducing impurities inorder to minimize chemical deterioration of the solubilized polypeptidedue to e.g. Maillard reaction. Scavenger molecules like glycyl glycineand ethylene diamine may be added to the formulations comprising polarorganic solvent (s) such as polyols to reduce deterioration of thepolypeptide whereas antioxidants can be added to reduce the rate offormation of further reducing impurities.

In one aspect of the invention, the polar organic solvent is present inthe pharmaceutical composition in an amount of 1-50% w/w, for example,5-40% w/w, for example, 5-30% w/w. Alternatively, the organic polarsolvent is present in an amount of 10-30% w/w, for example, 10-25% w/w,for example, in an amount of about 20% w/w or about 15% w/w.

For example, the polar organic polar solvent is propylene glycol and ispresent in the pharmaceutical composition in an amount of 1-50% w/w, forexample, 5-40% w/w, for example, 10-30% w/w, for example, 10-25% w/w,for example, 10-20% w/w, for example, about 20% w/w or about 15% w/w.

For example, the polar organic solvent is selected from the groupconsisting of glycerol, propylene glycol and mixtures thereof.

A solid hydrophilic component may be added to the pharmaceuticalcomposition in order to render or help render the pharmaceuticalcomposition solid or semi-solid at room temperature. The hydrophiliccomponent can comprise more than one excipient. If more than oneexcipient comprises the hydrophilic component, the hydrophilic componentcan be a mixture of liquids, solids, or both.

When a solid hydrophilic component is present, the pharmaceuticalcomposition may comprise from about 1% to about 25% by weight of solidhydrophilic component, e.g., from about 2% to about 20%, e.g., fromabout 3% to about 15%, e.g. from about 4% to about 10%.

An example of a hydrophilic component is PEG which is the polymer ofethylene oxide that conforms generally to the formula H(OCH₂CH₂)_(n)OHin which n correlates with the average molecular weight of the polymer.

The types of PEG useful in preparing pharmaceutical compositions can becategorized by its state of matter, i.e., whether the substance existsin a solid or liquid form at room temperature and pressure. As usedherein, “solid PEG” refers to PEG having a molecular weight such thatthe substance is in a solid state at room temperature and pressure. Forexample, PEG having a molecular weight ranging between 1,000 and 10,000is a solid PEG. Such PEGs include, but are not limited to PEG 1000, PEG1550, PEG 2000, PEG 3000, PEG 3350, PEG 4000 or PEG 8000. Particularlyuseful solid PEGs are those having a molecular weight between 1,450 and8,000. Especially useful as a solid

PEG are PEG 1450, PEG 3350, PEG 4000, PEG 8000, derivatives thereof andmixtures thereof. PEGs of various molecular weights arecommercially-available as the CARBOWAX SENTRY series from Dow Chemicals(Danbury, Conn.). Moreover, solid PEGs have a crystalline structure, orpolymeric matrix, Polyethylene oxide (“PEO”) which has an identicalstructure to PEG but for chain length and end groups are also suitable.Various grades of PEO are commercially available as POLYOX from DowChemicals. PEO, for example, has a molecular weight ranging from about100,000 to 7,000,000. The hydrophilic component can comprise PEG, PEO,and any combinations of the foregoing.

The hydrophilic components can optionally include a lower alkanol, e.g.,ethanol. While the use of ethanol is not essential, it can improvesolubility of the polypeptide in the carrier, improve storagecharacteristics and/or reduce the risk of drug precipitation.

In an alternative exemplary aspect, the hydrophilic component of thecarrier consists of a single hydrophilic component, e.g., a solid PEG,e.g., PEG 1450, PEG 3350, PEG 4000 and PEG 8000. In this exemplaryaspect, the hydrophilic phase of the microemulsion component consists ofa single hydrophilic substance. For example, if the carrier comprisedPEG 3350, the carrier would contain no other hydrophilic substances,e.g., lower alkanols (lower alkyl being C₁-C₄), such as ethanol; orwater.

In yet another alternative exemplary aspect, the hydrophilic componentof the carrier consists of a mixture of solid PEGs. For example, thehydrophilic component comprises PEG 1450, PEG 3350, PEG 4000, PEG 8000,derivatives thereof and any combinations and mixtures thereof.

In one aspect, the carrier comprises one or more surfactants, i.e.,optionally a mixture of surfactants; or surface active agents, whichreduce interfacial tension. The surfactant is, e.g., nonionic, ionic oramphoteric. Surfactants can be complex mixtures containing side productsor un-reacted starting products involved in the preparation thereof,e.g., surfactants made by polyoxyethylation may contain another sideproduct, e.g., PEG. The surfactant or surfactants have ahydrophilic-lipophilic balance (HLB) value which is at least 8. Forexample, the surfactant may have a mean HLB value of 8-30, e.g., 12-30,12-20 or 13-15. The surfactants can be liquid, semisolid or solid innature.

The term “surfactant” as used herein refers to any substance, inparticular a detergent that can adsorb at surfaces and interfaces, likeliquid to air, liquid to liquid, liquid to container or liquid to anysolid. The surfactant may be selected from a detergent, such asethoxylated castor oil, polyglycolyzed glycerides, acetylatedmonoglycerides, sorbitan fatty acid esters, polysorbate, such aspolysorbate-20, poloxamers, such as poloxamer 188 and poloxamer 407,polyoxyethylene sorbitan fatty acid esters, polyoxyethylene derivativessuch as alkylated and alkoxylated derivatives (tweens, e.g. Tween-20, orTween-80), monoglycerides or ethoxylated derivatives thereof,diglycerides or polyoxyethylene derivatives thereof, glycerol, cholicacid or derivatives thereof, lecithins, alcohols and phospholipids,glycerophospholipids (lecithins, cephalins, phosphatidyl serine),glyceroglycolipids (galactopyransoide), sphingophospholipids(sphingomyelin), and sphingoglycolipids (ceramides, gangliosides), DSS(docusate sodium, CAS registry no [577-11-7]), docusate calcium, CASregistry no [128-49-4]), docusate potassium, CAS registry no[7491-09-0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate),dipalmitoyl phosphatidic acid, sodium caprylate, bile acids and saltsthereof and glycine or taurine conjugates, ursodeoxycholic acid, sodiumcholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate,N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic(alkylarylsulphonates) monovalent surfactants, palmitoyllysophosphatidyl-L-serine, lysophospholipids (e.g.1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine), alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.,lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the postively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine,zwitterionic surfactants (e.g.N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate,dodecylphosphocholine, myristoyl lysophosphatidylcholine, hen egglysolecithin), cationic surfactants (quaternary ammonium bases) (e.g.cetyl-trimethylammonium bromide, cetylpyridinium chloride), non-ionicsurfactants (e.g., alkyl glucosides like dodecyl β-O-glucopyranoside,dodecyl β-O-maltoside, tetradecyl β-O-glucopyranoside, decylβ-O-maltoside, dodecyl β-O-maltoside, tetradecyl β-O-maltoside,hexadecyl β-D-maltoside, decyl β-D-maltotrioside, dodecylβ-D-maltotrioside, tetradecyl β-D-maltotrioside, hexadecyl13-D-maltotrioside, n-dodecyl-sucrose, n-decyl-sucrose, fatty alcoholethoxylates (e.g., polyoxyethylene alkyl ethers like octaethylene glycolmono tridecyl ether, octaethylene glycol mono dodecyl ether,octaethylene glycol mono tetradecyl ether), block copolymers aspolyethyleneoxide/polypropyleneoxide block copolymers(Pluronics/Tetronics, Triton X-100) ethoxylated sorbitan alkanoatessurfactants (e.g., Tween-40, Tween-80, Brij-35), fusidic acidderivatives (e.g., sodium tauro-dihydrofusidate etc.), longchain fattyacids and salts thereof C8-C20 (e.g., oleic acid and caprylic acid),acylcarnitines and derivatives, N-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, N-acylated derivatives of dipeptides comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, N-acylated derivative of a tripeptide comprising anycombination of a neutral amino acid and two charged amino acids, or thesurfactant may be selected from the group of imidazoline derivatives, ormixtures thereof.

Examples of solid surfactants include, but are not limited to,

1. reaction products of a natural or hydrogenated castor oil andethylene oxide. The natural or hydrogenated castor oil may be reactedwith ethylene oxide in a molar ratio of from about 1:35 to about 1:60,with optional removal of the PEG component from the products. Varioussuch surfactants are commercially available, e-g., the CREMOPHOR seriesfrom BASF Corp. (Mt. Olive, N.J.), such as CREMOPHOR RH 40 which isPEG40 hydrogenated castor oil which has a saponification value of about50- to 60, an acid value less than about one, a water content, i.e.,Fischer, less than about 2%, an n_(D) ⁶⁰ of about 1.453-1.457, and anHLB of about 14-16;

2. polyoxyethylene fatty acid esters that include polyoxyethylenestearic acid esters, such as the MYRJ series from Uniqema e.g., MYRJ 53having a m.p. of about 47° C.

Particular compounds in the MYRJ series are, e.g., MYRJ 53 having anm.p. of about 47° C. and PEG-40-stearate available as MYRJ 52;

3. sorbitan derivatives that include the TWEEN series from Uniqema,e.g., TWEEN 60;

4. polyoxyethylene-polyoxypropylene co-polymers and block co-polymers orpoloxamers, e.g., Pluronic F127, Pluronic F68 from BASF;

5. polyoxyethylene alkyl ethers, e.g., such as polyoxyethylene glycolethers of C₁₂-C₁₈ alcohols, e.g., polyoxyl 10- or 20-cetyl ether orpolyoxyl 23-lauryl ether, or 20-oleyl ether, or polyoxyl 10-, 20- or100-stearyl ether, as known and commercially available as the BRIJseries from Uniqema. Particularly useful products from the BRIJ seriesare BRIJ 58; BRIJ 76; BRIJ 78; BRIJ 35, i.e., polyoxyl 23 lauryl ether;and BRIJ 98, i.e., polyoxyl 20 oleyl ether. These products have a m.p.between about 32° C. to about 43° C.;

6. water-soluble tocopheryl PEG succinic acid esters available fromEastman Chemical Co. with a m.p. of about 36° C., e.g, TPGS, e.g.,vitamin E TPGS.

7. PEG sterol ethers having, e.g., from 5-35 [CH₂—CH, —O] units, e.g.,20-30 units, e-g., SO-LULAN C24 (Choleth-24 and Cetheth-24) from Chemron(Paso Robles, Calif.); similar products which may also be used are thosewhich are known and commercially available as NIKKOL BPS-30(polyethoxylated 30 phytosterol) and NIKKOL BPSH-25 (polyethoxylated 25phytostanol) from Nikko Chemicals;

8. polyglycerol fatty acid esters, e.g., having a range of glycerolunits from 4-10, or 4, 6 or 10 glycerol units. For example, particularlysuitable are deca-/hexa-/tetraglyceryl monostearate, e.g., DECAGLYN,HEXAGLYN and TETRAGLYN from Nikko Chemicals;

9. alkylene polyol ether or ester, e.g., lauroyl macrogol-32 glyceridesand/or stearoyl macrogol-32 glycerides which are GELUCIRE 44/14 andGELUCIRE 50/13 respectively;

10. polyoxyethylene mono esters of a saturated C₁₀ to C₂₂, such as C₁₈substituted e.g. hydroxy fatty acid; e.g. 12 hydroxy stearic acid PEGester, e.g. of PEG about e.g. 600-900 e.g. 660 Daltons MW, e.g. SOLUTOLHS 15 from BASF (Ludwigshafen, 20 Germany). According to a BASFtechnical leaflet MEF 151E (1986), SOLUTOL HS 15 comprises about 70%polyethoxylated 12-hydroxystearate by weight and about 30% by weightunesterified polyethylene glycol component. It has a hydrogenation valueof 90 to 110, a saponification value of 53 to 63, an acid number ofmaximum 1, and a maximum water content of 0.5% by weight;

11. polyoxyethylene-polyoxypropylene-alkyl ethers, e.g.polyoxyethylene-polyoxypropyleneethers of C₁₂ to C₁₈ alcohols, e.g.polyoxyethylen-20-polyoxypropylene-4-cetylether which is commerciallyavailable as NIKKOL PBC 34 from Nikko Chemicals;

12. polyethoxylated distearates, e.g. commercially available under thetradenames ATLAS G 1821 from Uniqema and NIKKOCDS-6000P from NikkoChemicals; and

13. lecithins, e.g., soy bean phospholipid, e.g. commercially availableas LIPOID S75 from Lipoid GmbH (Ludwigshafen, Germany) or eggphospholipid, commercially available as PHOSPHOLIPON 90 from NattermannPhospholipid (Cologne, Germany).

Examples of liquid surfactants include, but are not limited to, sorbitanderivatives such as TWEEN 20, TWEEN 40 and TWEEN 80, SYNPERONIC L44, andpolyoxyl 10-oleyl ether, all available from Uniqema, and polyoxyethylenecontaining surfactants e.g. PEG-8 caprylic/capric glycerides (e.g.Labrasol available from Gattefosse).

The composition of the invention may comprise from about 0% to about 95%by weight surfactant, e.g. from about 5% to about 80% by weight, e.g.,about 10% to about 70% by weight, e.g., from about 20% to about 60% byweight, e.g., from about 30% to about 50%.

In one aspect, the surfactant is polyoxyethylene-polyoxypropyleneco-polymers and block co-polymers or poloxamers, e.g., Pluronic F127,Pluronic F68 from BASF.

In one aspect, the surfactant is a poloxamer. In a further aspect, thesurfactant is selected from the group consisting of poloxamer 188,poloxamer 407 and mixtures of poloxamer 407 and poloxamer 188.

In one aspect, the surfactant is a polyoxyethylene containingsurfactants e.g., PEG-8 caprylic/capric glycerides (e.g., Labrasolavailable from Gattefosse).

In one aspect, the surfactant is a lauroyl polyoxylglyceride (e.g.Gelucire 44/14 available from Gattefosse).

In one aspect, the surfactant is Cremophor RH40 from BASF.

In certain aspects, the pharmaceutical composition may compriseadditional excipients commonly found in pharmaceutical compositions,examples of such excipients include, but are not limited to,antioxidants, antimicrobial agents, enzyme inhibitors, stabilizers,preservatives, flavors, sweeteners and other components as described inHandbook of Pharmaceutical Excipients, Rowe et al., Eds., 4'h Edition,Pharmaceutical Press (2003), which is hereby incorporated by reference.

These additional excipients may be in an amount from about 0.05-5% byweight of the total pharmaceutical composition. Antioxidants,anti-microbial agents, enzyme inhibitors, stabilizers or preservativestypically provide up to about 0.05-1% by weight of the totalpharmaceutical composition. Sweetening or flavoring agents typicallyprovide up to about 2.5% or 5% by weight of the total pharmaceuticalcomposition.

Examples of antioxidants include, but are not limited to, ascorbic acidand its derivatives, tocopherol and its derivatives, butyl hydroxylanisole and butyl hydroxyl toluene.

In one aspect, the composition comprises a buffer. The term “buffer” asused herein refers to a chemical compound in a pharmaceuticalcomposition that reduces the tendency of pH of the composition to changeover time as would otherwise occur due to chemical reactions. Buffersinclude chemicals such as sodium phosphate, TRIS, glycine and sodiumcitrate.

The term “preservative” as used herein refers to a chemical compoundwhich is added to a pharmaceutical composition to prevent or delaymicrobial activity (growth and metabolism). Examples of pharmaceuticallyacceptable preservatives are phenol, m-cresol and a mixture of phenoland m-cresol.

The term “stabilizer” as used herein refers to chemicals added topeptide containing pharmaceutical compositions in order to stabilize thepeptide, i.e., to increase the shelf life and/or in-use time of suchcompositions. Examples of stabilizers used in pharmaceuticalformulations are L-glycine, L-histidine, arginine, glycylglycine,ethylenediamine, citrate, EDTA, zinc, sodium chloride, polyethyleneglycol, carboxymethylcellulose, and surfactants and antioxidants likealfa-tocopherol and I-ascorbic acid.

In a further aspect, a process for preparing a pharmaceuticalcomposition, containing an acylated protease stabilised insulin,comprises the steps of bringing the drug and a carrier comprising apolar organic solvent, a lipophilic component, and optionally asurfactant and/or a hydrophilic component into intimate admixture. Forexample, the acylated protease stabilised insulin and the carrier can beliquefied, for example, by heating to about 20° C. to about 80° C., andthen solidifying by cooling to room temperature.

The carrier can be prepared separately before bringing a carriercomprising a polar organic solvent, a lipophilic component, andoptionally a surfactant and/or a hydrophilic component into intimateadmixture with the derivatized insulin peptide. Alternatively, one, twoor more of the components of the carrier can be mixed together with thepolypeptide.

The acylated protease stabilised insulin can be dissolved in the polarorganic solvent, and then be mixed with the lipid component andoptionally with a surfactant.

Alternatively, a process for preparing a pharmaceutical composition suchas SEDDS or SMEDDS (which can be filled into a capsule, e.g. entericcoated capsule, soft capsule, enteric soft capsule) containing anacylated protease stabilised insulin, comprises the following steps:

(a) dissolving the derivatized insulin peptide in the polar organicsolvent and

(b) mixing with the lipophilic component, surfactant and optionallyhydrophilic component.

For example, a process for preparing the pharmaceutical composition iscarried out at low temperature (e.g. room temperature or below roomtemperature).

When preparing the pharmaceutical composition, the acylated proteasestabilised insulin may, e.g., be dissolved in the polar organic solventusing the following method:

a) providing an aqueous solution of the acylated protease stabilisedinsulin, optionally comprising excipients,

b) adjusting the pH value to a target pH value which is 1 unit,alternatively 2 units and alternatively 2.5 pH units above or below thepl of the acylated protease stabilised insulin,

c) removing water (dehydrating) from the acylated protease stabilisedinsulin by conventional drying technologies such as freeze- and spraydrying, and

d) mixing and dissolution of the acylated protease stabilised insulin insaid polar non-aqueous solvent, e.g., by stirring, tumbling or othermixing methods,

e) optionally filtration or centrifugation of the non-aqueous solutionof the acylated protease stabilised insulin to remove non-dissolvedinorganic salts,

f) optionally removing residual amounts of waters by, e.g., adding soliddessicants or vacuum drying.

For example, the acylated protease stabilised insulin is dissolved inthe polar organic solvent by the following method:

a) providing an aqueous solution of a acylated protease stabilisedinsulin, optionally containing stabilizers such as zinc andglycylglycine,

b) adjusting the pH value to 1 unit, alternatively 2 units andalternatively 2.5 pH units above or below the pl of the polypeptide,e.g., by adding a non-volatile base or a acid, such as hydrochloric acidor sodium hydroxide, to the solution,

c) removing water (dehydrating) from the acylated protease stabilisedinsulin by conventional drying technologies such as freeze- and spraydrying,

d) mixing and dissolution of the acylated protease stabilised insulin insaid polar non-aqueous solvent, e.g., by stirring, tumbling or othermixing methods,

e) optionally filtration or centrifugation of the non-aqueous solutionof the acylated protease stabilised insulin to remove non-dissolvedinorganic salts,

f) optionally removing residual amounts of waters by, e.g., adding soliddessicants or vacuum drying.

By “volatile base” is meant a base, which to some extend will evaporateupon heating and/or at reduced pressure, e.g., bases which have a vapourpressure above 65 Pa at room temperature or an aqueous azeotropicmixture including a base having a vapour pressure above 65 Pa at roomtemperature. Examples of volatile bases are ammonium hydroxides,tetraalkylammonium hydroxides, secondary amines, tertiary amines, arylamines, alphatic amines or ammonium bicarbonate or a combination. Forexample the volatile base can be bicarbonate, carbonate, ammonia,hydrazine or an organic base such as a lower aliphatic amines e.g.trimethyl amine, triethylamine, diethanolamines, triethanolamine andtheir salts. Furthermore, the volatile base can be ammonium hydroxide,ethyl amine or methyl amine or a combination hereof.

By “volatile acid” is meant an acid, which to some extend will evaporateupon heating and/or at reduced pressure, e.g., acids which have a vapourpressure above 65 Pa at room temperature or an aqueous azeotropicmixture including an acid having a vapour pressure above 65 Pa at roomtemperature. Examples of volatile acids are carbonic acid, formic acid,acetic acid, propionic acid and butyric acid.

A “non volatile base” as mentioned herein means a base, which do notevaporate or only partly evaporate upon heating, e.g., bases with avapour pressure below 65 Pa at room temperature. The non volatile basecan be selected from the group consisting of alkaline metal salts,alkaline metal hydroxides, alkaline earth metal salts, alkaline earthmetal hydroxides and amino acids or a combination hereof. Examples ofnon-volatile bases are sodium hydroxide, potassium hydroxide, calciumhydroxide, and calcium oxide.

A “non volatile acid” as mentioned herein means an acid, which do notevaporate or only partly evaporate upon heating, e.g., bases with avapour pressure below 65 Pa at room temperature. Examples ofnon-volatile acids are hydrochloric acid, phosphoric acid and sulfuricacid.

The acylated protease stabilised insulin may be present in an amount upto about 40% such as up to about 20% by weight of the composition, orfrom about 0.01% such as from about 0.1%, alternatively, from about0.01% to about 20%, alternatively, from about 1% to 20% or from about 1%to 10% by weight of the composition. It is intended, however, that thechoice of a particular level of polypeptide will be made in accordancewith factors well-known in the pharmaceutical arts, including thesolubility of the polypeptide in the polar organic solvent or optionalhydrophilic component or surfactant used, or a mixture thereof, mode ofadministration and the size and condition of the patient.

For example, the pharmaceutical formulation comprises an acylatedprotease stabilised insulin in a concentration from 0.1% w/w to 30% w/w.

Each unit dosage will suitably contain from 0.1 mg to 300 mg acylatedprotease stabilised insulin polypeptide, e.g., about 0.1 mg, 1 mg, 5 mg,10 mg, 15 mg, 25 mg, 50 mg, 100 mg, 200 mg, 250 mg, 300 mg, e.g.,between 5 mg and 300 mg of the acylated protease stabilised insulin. Forexample, each unit dosage contains between 10 mg and 300 mg, for example10 mg and 100 mg or between 20 mg and 300 mg, fore example, between 20mg and 100 mg of the acylated protease stabilised insulin. Such unitdosage forms are suitable for administration 1-5 times daily dependingupon the particular purpose of therapy.

The acylated protease stabilsed insulin is pH optimized beforedissolution in the polar organic solvent to improve solubility in thepolar organic solvent.

When using the term “pH optimized” it is herein meant that the acylatedprotease stabilsed insulin has been dehydrated at a target pH which isat least 1 pH unit from the pl of the acylated protease stabilsedinsulin in aqueous solution. Thus, the target pH is more than 1 pH unitabove the isoelectric point of the acylated protease stabilsed insulin.Alternatively, the target pH is more than 1 pH unit below theisoelectric point of the acylated protease stabilsed insulin. Hence, thetarget pH could be more than 1.5 pH units above or below the pl, forexample, 2.0 pH units or more above or below the pl, for example, 2.5 pHunits or more above or below the pl of the acylated protease stabilsedinsulin.

The term “dehydrated” as used herein in connection with an acylatedprotease stabilsed insulin refers to a derivatized acylated proteasestabilsed insulin which has been dried from an aqueous solution. Theterm “target pH” as used herein refers to the aqueous pH which willestablish when the dehydrated acylated protease stabilsed insulin isrehydrated in pure water to a concentration of approximately 40 mg/ml ormore. The target pH will typically be identical to the pH of the aqueoussolution of the acylated protease stabilsed insulin from which theacylated protease stabilsed insulin was recovered by drying. However,the pH of the acylated protease stabilsed insulin solution will not beidentical to the target pH, if the solution contains volatile acids orbases. It has been found that the pH history of the acylated proteasestabilsed insulin will be determinant for the amount of the acylatedprotease stabilsed insulin, which can be solubilized in the polarorganic solvent.

The term “the pl of the polypeptide” as used herein refers to theisoelectric point of a polypeptide.

The term “isoelectric point” as used herein means the pH value where theoverall net charge of a macromolecule such as a peptide is zero. Inpeptides there may be several charged groups, and at the isoelectricpoint the sum of all these charges is zero. At a pH above theisoelectric point the overall net charge of the peptide will benegative, whereas at pH values below the isoelectric point the overallnet charge of the peptide will be positive.

The pl of a protein can be determined experimentally by electrophoresistechniques such as electrofocusing:

A pH gradient is established in an anticonvective medium, such as apolyacrylamide gel. When a protein is introduced in to the system itwill migrate under influence of an electric field applied across thegel. Positive charged proteins will migrate to the cathode. Eventually,the migrating protein reaches a point in the pH gradient where its netelectrical charge is zero and is said to be focused. This is theisoelectric pH (pl) of the protein. The protein is then fixed on the geland stained. The pl of the protein can then be determined by comparisonof the position of the protein on the gel relative to marker moleculeswith known pl values.

The net charge of a protein at a given pH value can be estimatedtheoretically by a person skilled in the art by conventional methods. Inessence, the net charge of protein is the equivalent to the sum of thefractional charges of the charged amino acids in the protein: aspartate(β-carboxyl group), glutamate (δ-carboxyl group), cysteine (thiolgroup), tyrosine (phenol group), histidine (imidazole side chains),lysine (∈-ammonium group) and arginine (guanidinium group).Additionally, one should also take into account charge of proteinterminal groups (α-NH₂ and α-COOH). The fractional charge of theionisable groups can be calculated from the intrinsic pKa values.

The drying, i.e., dehydration of the acylated protease stabilsed insulincan be performed by any conventional drying method such, e.g., byspray-, freeze-, vacuum-, open- and contact drying. For example, theacylated protease stabilsed insulin solution is spray dried to obtain awater content below about 10%, for example, below about 8%, below about6%, below about 5%, below about 4%, below about 3%, below about 2% orbelow about 1% calculated on/measured by loss on drying test(gravimetric) as stated in the experimental part.

Fore example, the acylated protease stabilised insulin is spray dried orfreeze-dried.

Compositions containing acylated protease stabilised insulins of thisinvention can be used in the treatment of states which are sensitive toinsulin. Thus, they can be used in the treatment of type 1 diabetes,type 2 diabetes and hyperglycaemia for example as sometimes seen inseriously injured persons and persons who have undergone major surgery.The optimal dose level for any patient will depend on a variety offactors including the efficacy of the specific insulin derivativeemployed, the age, body weight, physical activity, and diet of thepatient, on a possible combination with other drugs, and on the severityof the state to be treated. It is recommended that the daily dosage ofthe acylated insulin of this invention be determined for each individualpatient by those skilled in the art in a similar way as for knowninsulin compositions.

Preferred Features of this Invention

The features of this invention are as follows:

-   1. An acylated protease stabilised insulin wherein the protease    stabilised insulin, formally, consists of a non-protease stabilised    insulin (parent insulin) wherein at least one hydrophobic amino acid    has been substituted with hydrophilic amino acids, and wherein said    substitution is within or in close proximity to one or more protease    cleavage sites of the non-protease stabilised insulin (parent    insulin) and wherein such protease stabilised insulin optionally    further comprises one or more additional mutations with the proviso    that there is only one lysine residue in the stabilized insulin, and    wherein the acyl moiety is attached to the lysine residue or to a    N-terminal position in the protease stabilized insulin.-   2. An acylated protease stabilised insulin wherein the protease    stabilised insulin, formally, consists of a non-protease stabilised    insulin (parent insulin) wherein at least two hydrophobic amino    acids have been substituted with hydrophilic amino acids, and    wherein said substitutions are within or in close proximity to two    or more protease cleavage sites of the non-protease stabilised    insulin (parent insulin) and wherein such protease stabilised    insulin optionally further comprises one or more additional    mutations with the proviso that there is only one lysine residue in    the stabilized insulin, and wherein the acyl moiety is attached to    the lysine residue in the protease stabilized insulin.-   3. An acylated protease stabilised insulin wherein the protease    stabilised insulin, formally, consists of a non-protease stabilised    insulin (parent insulin) wherein at least two hydrophobic amino    acids have been substituted with hydrophilic amino acids, and    wherein said substitutions are within or in close proximity to two    or more protease cleavage sites of the non-protease stabilised    insulin (parent insulin) and wherein such protease stabilised    insulin optionally further comprises one or more additional    mutations with the proviso that there is only one lysine residue in    the stabilized insulin, and wherein the acyl moiety is attached to    the lysine residue or to a N-terminal position in the protease    stabilized insulin.-   4. An acylated insulin according any of the preceding clauses    wherein the protease stabilised insulin has increased solubility    relative to the acylated parent insulin.-   5. An acylated insulin according to any one of the preceding clauses    to the extent possible wherein the B-chain of the insulin comprises    at least one mutation relative to the parent insulin.-   6. An acylated insulin according to the preceding clause to the    extent possible wherein the B-chain of the insulin comprises one,    two or three but not more mutations relative to the parent insulin.-   7. An acylated insluin according to any one of the preceding clauses    to the extent possible, wherein the A chain of the protease    stabilised insulin is identical with the A chain of human insulin.-   8. An acylated insulin according to any one of the preceding clauses    to the extend possible wherein the A-chain of the insulin comprises    at least one mutation and the B-chain of the insulin comprises at    least one mutation relative to the parent insulin.-   9. An acylated insulin according to any one of the preceding clauses    to the extend possible wherein the A-chain of the insulin comprises    at least two mutations and the B-chain of the insulin comprises at    least one mutation relative to the parent insulin.-   10. An acylated insulin according to any one of the preceding    clauses to the extent possible wherein the insulin further comprises    at least one amino acid substitution in a protease site of a first    modified protease stabilised insulin, wherein said at least one    amino acid substitution is such that at least one hydrophobic amino    acid has been substituted with at least one hydrophilic amino acid.-   11. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the amino acid in    position A12 is Glu or Asp; and/or the amino acid in position A13 is    His, Asn, Glu or Asp; and/or the amino acid in position A14 is Tyr,    Asn, Gln, Glu, Arg, Asp, Gly or His; and/or the amino acid in    position A15 is Glu or Asp; and the amino acid in position B24 is    His; and/or the amino acid in position B25 is His or Asn; and/or the    amino acid in position B26 is His, Gly, Asp or Thr; and/or the amino    acid in position B27 is His, Glu, Asp, Gly or Arg; and/or the amino    acid in position B28 is His, Gly, Glu or Asp; and which optionally    further comprises one or more additional mutations.-   12. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the amino acid in    position A12 is Glu or Asp; and/or the amino acid in position A13 is    His, Asn, Glu or Asp; and/or the amino acid in position A14 is Tyr,    Asn, Gln, Glu, Arg, Asp, Gly or His; and/or the amino acid in    position A15 is Glu or Asp; and/or the amino acid in position B16 is    Tyr, His or Glu; and/or the amino acid in position B24 is His;    and/or the amino acid in position B25 is His or Asn; and/or the    amino acid in position B26 is His, Gly, Asp or Thr; and/or the amino    acid in position B27 is His, Glu, Asp, Gly, Lys, Arg or deleted;    and/or the amino acid in position B28 is His, Gly, Glu, Asp, or    absent (deleted); and/or the amino acid in position B29 is Lys, Arg,    or absent (deleted); and which optionally further comprises one or    more additional mutations and, preferably, acylated protease    stabilised insulins wherein the amino acid in position A12 is Glu or    Asp; and/or the amino acid in position A13 is His, Asn, Glu or Asp;    and/or the amino acid in position A14 is Tyr, Asn, Gln, Glu, Arg,    Asp, Gly or His; and/or the amino acid in position A15 is Glu or    Asp; and the amino acid in position B24 is His; and/or the amino    acid in position B25 is His or Asn; and/or the amino acid in    position B26 is His, Gly, Asp or Thr; and/or the amino acid in    position B27 is His, Glu, Asp, Gly or Arg; and/or the amino acid in    position B28 is His, Gly, Glu, or Asp; and which optionally further    comprises one or more additional mutations.-   13. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the amino acid in    position A14 is Glu, Asp or His, the amino acid in position B25 is    His or Asn and which optionally further comprises one or more    additional mutations.-   14. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the amino acid in    position A14 is Glu, Asp or His, the amino acid in position B25 is    His or Asn and the amino acid in position B30 is deleted.-   15. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the amino acid in    position A14 is Glu, Asp or His, the amino acid in position B16 is    His or Glu, the amino acid in position B25 is His and the amino acid    in position B30 is deleted.-   16. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the amino acid in    position A14 is Glu, Asp or His and the amino acid in position B25    is His and the amino acid in position B30 is deleted.-   17. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the amino acid in    position A14 is Glu or Asp and the amino acid in position B28 is Glu    or Asp, and, optionally, there is no amino acid residue in the B30    position.-   18. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the one or more    additional mutations is selected from a group consisting of: A8His,    A18Gln, A21Gln, A21Gly, B1Glu, B1Gln, B3Gln, B10Pro, B14Thr, B16Glu,    B17Ser, B26Asp, B27Glu, B27Asp, B28Asp, B28Glu, and desB30.-   19. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the additional    mutation is desB30.-   20. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein A14 is Glu.-   21. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein B25 is Asn.-   22. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein B25 is His.-   23. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein B25 is Asn and B27    is Glu or Asp.-   24. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein B25 is Asn and B27    is Glu.-   25. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible which shows increased    stability towards one or more protease enzymes relative to the    parent protein.-   26. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible which shows increased    stability towards two or more protease enzymes relative to the    parent protein.-   27. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the parent insulin    is selected from a group consisting of a) human insulin; b) an    insulin analogue of human insulin wherein the amino acid residue in    position B28 is Pro, Asp, Lys, Leu, Val or Ala and the amino acid    residue in position B29 is Lys or Pro and optionally the amino acid    residue in position B30 is deleted; c) des(B26-B30) human insulin,    des(B27-B30) human insulin, des(B28-B30) human insulin, des(B29-B30)    human insulin, des(B27) human insulin or des(B30) human insulin; d)    an insulin analogue of human insulin wherein the amino acid residue    in position B3 is Lys and the amino acid residue in position B29 is    Glu or Asp; e) an insulin analogue of human insulin wherein the    amino acid residue in position A21 is Gly and wherein the insulin    analogue is further extended in the C-terminal with two Arg    residues; f) an insulin derivative wherein the amino acid residue in    position B30 is substituted with a threonine methyl ester; and g) an    insulin derivative wherein to the NE position of lysine in the    position B29 of des(B30) human insulin a tetradecanoyl chain is    attached.-   28. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the one or more    additional mutations are selected to enhance chemical stability of    insulin.-   29. An acylated protease stabilised insulin according to the    preceding clause to the extent possible wherein the one or more    additional mutations are selected from a group consisting of A18Gln,    A21Gln, A21 Gly and B3Gln.-   30. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible comprising an A-chain amino    acid sequence of formula 1, i.e.:    Xaa⁽⁻²⁾-Xaa_(A(−1))-Xaa_(A0)-Gly-Ile-Val-Glu-Gln-Cys-Cys-Xaa_(A8)-Ser-Ile-Cys-Xaa_(A12)-Xaa_(A13)-Xaa_(A14)-Xaa_(A18)-Leu-Glu-Xaa_(A18)-Tyr-Cys-Xaa_(A21)    (SEQ ID No:1), and a B-chain amino acid sequence of formula 2, i.e.:    Xaa_(B(−2))-Xaa_(B(−1))-Xaa_(B0)-Xaa_(B1)-Xaa_(B2)-Xaa_(B3)-Xaa_(B4)-His-Leu-Cys-Gly-Ser-Xaa_(B10)-Leu-Val-Glu-Ala-Leu-Xaa_(B18)-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa_(B24)-Xaa_(B25)-Xaa_(B26)-Xaa_(B27)-Xaa_(B28)-Xaa_(B29)-Xaa_(B30)-Xaa_(B31)-Xaa_(B32)    (SEQ ID No:2), wherein Xaa_(k2)) is absent or Gly; Xaa⁽⁻¹⁾ is absent    or Pro; Xaa_(A0) is absent or Pro; Xaa_(A8) is independently    selected from Thr and His; Xaa_(A12) is independently selected from    Ser, Asp and Glu; Xaa_(A13) is independently selected from Leu, Thr,    Asn, Asp, Gln, His, Lys, Gly, Arg, Pro, Ser and Glu; Xaa_(A14) is    independently selected from Tyr, Thr, Asn, Asp, Gln, His, Lys, Gly,    Arg, Pro, Ser and Glu; Xaa_(A15) is independently selected from Gln,    Asp and Glu; Xaa_(A18) is independently selected from Asn, Lys and    Gln; Xaa_(A21) is independently selected from Asn and Gln;    Xaa_(B(−2)) is absent or Gly; Xaa_(B(−1)) is absent or Pro; Xaa_(B0)    is absent or Pro; Xaa_(B1) is absent or independently selected from    Phe and Glu; Xaa_(B2) is absent or Val; Xaa_(B3) is absent or    independently selected from Asn and Gln; Xaa_(B4) is independently    selected from Gln and Glu; Xaa_(B10) is independently selected from    His, Asp, Pro and Glu; Xaa_(B16) is independently selected from Tyr,    Asp, Gln, His, Arg, and Glu; Xaa_(B24) is independently selected    from Phe and His; Xaa_(B25) is independently selected from Phe, Asn    and His; Xaa_(B26) is absent or independently selected from Tyr,    His, Thr, Gly and Asp; Xaa_(B27) is absent or independently selected    from Thr, Asn, Asp, Gln, His, Gly, Arg, Pro, Ser and Glu; Xaa_(B28)    is absent or independently selected from Pro, His, Gly and Asp;    Xaa_(B29) is absent or independently selected from Lys and Gln;    Xaa_(B30) is absent or Thr; Xaa_(B31) is absent or Leu; Xaa_(B32) is    absent or Glu; the C-terminal may optionally be derivatized as an    amide; wherein the A-chain amino acid sequence and the B-chain amino    acid sequence are connected by disulphide bridges between the    cysteines in position 7 of the A-chain and the cysteine in position    7 of the B-chain, and between the cysteine in position 20 of the    A-chain and the cysteine in position 19 of the B-chain and wherein    the cysteines in position 6 and 11 of the A-chain are connected by a    disulphide bridge; wherein optionally the N-terminal A-chain amino    acid sequence is connected to the C-terminal B-chain amino acid    sequence by an amino acid sequence comprising 3-7 amino acids to    form a single chain insulin molecule, wherein optionally the    N-terminal of the B-chain is extended with 1-10 amino acids; wherein    if Xaa_(A8) is Thr and Xaa_(A12) is Ser and Xaa_(A13) is Leu and    Xaa_(A14) is Tyr then Xaa_(A15) is Glu or Asp; and wherein if    Xaa_(B24) is Phe and Xaa_(B25) is Phe and Xaa_(B26) is Tyr and    Xaa_(B27) is Thr and Xaa_(B28) is Pro then Xaa_(B29) Gln.-   31. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible comprising an A-chain amino    acid sequence of formula 3, i.e.:    Gly-Ile-Val-Glu-Gln-Cys-Cys-Xaa_(A8)-Ser-Ile-Cys-Xaa_(A12)-Xaa_(A13)-Xaa_(A14)-Xaa_(A15)-Leu-Glu-Xaa_(A18)-Tyr-Cys-Xaa_(A21)    (SEQ ID No:3), and a B-chain amino acid sequence of formula 4, i.e.:    Xaa_(B1)-Val-Xaa_(B3)-Xaa_(B4)-His-Leu-Cys-Gly-Ser-Xaa_(B10)-Leu-Val-Glu-Ala-Leu-Xaa_(B16)-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa_(B24)-His-Xaa_(B26)-Xaa_(B27)-Xaa_(B28)-Xaa_(B29)-Xaa_(B30)    (SEQ ID No:4), wherein Xaa_(A8) is independently selected from Thr    and His; Xaa_(A12) is independently selected from Ser, Asp and Glu;    Xaa_(A13) is independently selected from Leu, Thr, Asn, Asp, Gln,    His, Lys, Gly, Arg, Pro, Ser and Glu; Xaa_(A14) is independently    selected from Tyr, Thr, Asn, Asp, Gln, His, Lys, Gly, Arg, Pro, Ser    and Glu; Xaa_(A15) is independently selected from Gln, Asp and Glu;    Xaa_(A18) is independently selected from Asn, Lys and Gln; Xaa_(A21)    is independently selected from Asn, and Gln; Xaa_(B1) is    independently selected from Phe and Glu; Xaa_(B3) is independently    selected from Asn and Gln; Xaa_(B4) is independently selected from    Gln and Glu; Xaa_(B10) is independently selected from His, Asp, Pro    and Glu; Xaa_(B16) is independently selected from Tyr, Asp, Gln,    His, Arg, and Glu; Xaa_(B24) is independently selected from Phe and    His; Xaa_(B25) is independently selected from Phe, Asn and His;    Xaa_(B26) is absent or independently selected from Tyr, His, Thr,    Gly and Asp; Xaa_(B27) is absent or independently selected from Thr,    Asn, Asp, Gln, His, Gly, Arg, Pro, Ser and Glu; Xaa_(B28) is absent    or independently selected from Pro, His, Gly and Asp; Xaa_(B29) is    absent or independently selected from Lys and Gln; Xaa_(B30) is    absent or Thr; the C-terminal may optionally be derivatized as an    amide; wherein the A-chain amino acid sequence and the B-chain amino    acid sequence are connected by disulphide bridges between the    cysteines in position 7 of the A-chain and the cysteine in position    7 of the B-chain, and between the cysteine in position 20 of the    A-chain and the cysteine in position 19 of the B-chain and wherein    the cysteines in position 6 and 11 of the A-chain are connected by a    disulphide bridge.-   32. An acylated protease stabilised insulin according to the    preceding clause to the extent possible, wherein Xaa_(A8) is    independently selected from Thr and His; Xaa_(A12) is independently    selected from Ser and Glu; Xaa_(A13) is independently selected from    Leu, Thr, Asn, Asp, Gln, His, Lys, Gly, Arg, Pro, Ser and Glu;    Xaa_(A14) is independently selected from Tyr, Asp, His, and Glu;    Xaa_(A15) is independently selected from Gln and Glu; Xaa_(A18) is    independently selected from Asn, Lys and Gln; Xaa_(A21) is    independently selected from Asn, and Gln; Xaa_(B1) is independently    selected from Phe and Glu; Xaa_(B3) is independently selected from    Asn and Gln; Xaa_(B4) is independently selected from Gln and Glu;    Xaa_(B10) is independently selected from His, Asp, Pro and Glu;    Xaa_(B16) is independently selected from Tyr, Asp, Gln, His, Arg,    and Glu; Xaa_(B24) is independently selected from Phe and His;    Xaa_(B25) is independently selected from Phe, Asn and His; Xaa_(B26)    is independently selected from Tyr, Thr, Gly and Asp; Xaa_(B27) is    independently selected from Thr, Asn, Asp, Gln, His, Lys, Gly, Arg,    and Glu; Xaa_(B28) is independently selected from Pro, Gly and Asp;    Xaa_(B29) is independently selected from Lys and Gln; Xaa_(B30) is    absent or Thr; the C-terminal may optionally be derivatized as an    amide; wherein the A-chain amino acid sequence and the B-chain amino    acid sequence are connected by disulphide bridges between the    cysteines in position 7 of the A-chain and the cysteine in position    7 of the B-chain, and between the cysteine in position 20 of the    A-chain and the cysteine in position 19 of the B-chain and wherein    the cysteines in position 6 and 11 of the A-chain are connected by a    disulphide bridge.-   33. An acylated protease stabilised insulin wherein, in the protease    stabilised insulin, the amino acid in position A14 is Glu or His    (i.e., E or H, according to the one letter code), the amino acid in    position B25 is His and which optionally further comprises one or    more additional mutations, and wherein the acyl moiety is attached    to the ∈ amino group in the lysine residue in position B29.-   34. An acylated protease stabilised insulin wherein, in the protease    stabilised insulin, the amino acid in position B25 is His or Asn,    the amino acid in position B27 is Glu or Asp, and which optionally    further comprises one or more of the following additional mutations:    A8H, A14E/D, B1E/D, B28E/D, and desB30 and wherein the acyl moiety    is attached to the ∈ amino group in the lysine residue in position    B29.-   35. An acylated protease stabilised insulin wherein, in the protease    stabilised insulin, the amino acid in position A14 is Tyr, Glu or    His (i.e., Y, E or H, according to the one letter code), the amino    acid in position B25 is Asn, the amino acid in position B27 is Glu    or Asp and which optionally further comprises one or more additional    mutations, and wherein the acyl moiety is attached to the ∈ amino    group in the lysine residue in position B29.-   36. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein the protease    stabilised insulin comprises the A14E mutation.-   37. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein, in the    protease stabilised insulin, apart from the mutation in position    B25, there is only the mutation in position A14 mentioned in the    preceding clause.-   38. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein the protease    stabilised insulin comprises the A14H mutation.-   39. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein the protease    stabilised insulin analogue comprises the desB30 mutation.-   40. An acylated protease stabilised insulin according to any of the    preceding clauses to the extent possible wherein the one or more    additional mutations within the protease stabilised insulin is    selected from a group consisting of: A(−1)P, A(O)P, A8H, A21G,    B(−1)P, B(O)P, B1E, B10, B16E, B26D, B27E, B28D, desB30, B31L and    B32E.-   41. An acylated protease stabilised insulin according to the    preceding clause to the extent possible, wherein the protease    stabilised insulin, apart from the mutations in positions A14 and    B25, has only one of the mutations mentioned in the previous    clauses.-   42. An acylated protease stabilised insulin according to any one of    the preceding clauses but the last one (i.e., except clause 41) to    the extent possible, wherein the protease stabilised insulin, apart    from the mutations in positions A14 and B25, has exactly two of the    mutations mentioned in the preceding clause but two (i.e., mentioned    in clause 40).-   43. An acylated protease stabilised insulin according to any one of    the preceding clauses but the last two (i.e. except clauses 41    and 42) to the extent possible, wherein the protease stabilised    insulin, apart from the mutations in positions A14 and B25, has    exactly three of the mutations mentioned in the preceding clause but    two (i.e., mentioned in clause 40).-   44. An acylated protease stabilised insulin according to any one of    the preceding clauses but the last two (i.e. except clauses 41    and 42) to the extent possible wherein, apart from the mutations in    positions A14 and B25, the only additional mutation is desB30.-   45. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein the C terminal    amino acid residue in the A chain of the protease stabilized insulin    is the A21 amino acid residue.-   46. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein the protease    stabilized insulin is selected from the group consisting of A8H,    B25N, B27E, desB30 human insulin; A14E, A18L, B25H, desB30 human    insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, B1E,    B25H, B27E, B28E, desB30 human insulin; A14E, B1E, B25H, B28E,    desB30 human insulin; A14E, B1E, B27E, B28E, desB30 human insulin;    A14E, B1E, B28E, desB30 human insulin; A14E, B16H, B25H, desB30    human insulin; A14E, B25H, desB30 human insulin; A14E, B25H, B26G,    B27G, B28G, desB30 human insulin; A14E, B25H, B27E, desB30 human    insulin; A14E, B25H, desB27, desB30 human insulin; A14E, B25H, B29R,    desB30 human insulin; A14E, B28D, desB30 human insulin; A14E, B28E,    desB30 human insulin; B25N, B27E, desB30 human insulin; B25H, desB30    human insulin; A14E, B25H, B26G, B27G, B28G, B29R, desB30 human    insulin; A14E, B25H, B29R, desB30 human insulin; A14E, A21G, B25H,    desB27, desB30 human insulin; A14E, A21G, B25H, desB30 human    insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B25H, B16H,    desB30 human insulin; A14E, B25H, B26G, B27G, B28G, desB30 human    insulin; A14E, B25H, desB27, desB30 human insulin; A14E, B25H, B27K,    desB28, desB29, desB30 human insulin; A14E, B25H, desB30 human    insulin; A14E, desB30 human insulin and A21G, B25H, desB30 human    insulin.-   47. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein the acyl moiety    attached to the protease stabilised insulin has the general formula    Acy-AA1_(n)-AA2_(m)-AA3_(p)-(I), wherein Acy, AA1, AA2, AA3, n, m    and p are as defined above.-   48. An acylated protease stabilised insulin according to the    preceding clause to the extent possible wherein Acy is a fatty acid,    preferably myristic acid or steric acid, more preferred myristic    acid.-   49. An acylated protease stabilised insulin according to any one of    the preceding clauses except the last one, wherein Acy is a fatty    diacid, preferably a fatty (α,ω) diacid, more preferred    heptadecanedioic acid, hexadecanedioic acid, octadecanedioic acid,    nonadecanedioic acid, docosanedioic acid, eicosanedioic acid.-   50. An acylated protease stabilised insulin according to any one of    the preceding clauses except the last one, wherein Acy is a    ω-(tetrazol-5-yl)-fatty acid, preferably    15-(1H-tetrazol-5-yl)pentadecanoic acid,    16-(1H-tetrazol-5-yl)hexadecanoic acid,    17-(1H-tetrazol-5-yl)heptadecanoic acid,    18-(1H-tetrazol-5-yl)octadecanoic acid, or    19-(1H-tetrazol-5-yl)nonadecanoic acid.-   51. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein AA1 is    tranexamic acid or glycine.-   52. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein AA1 is    tranexamic acid.-   53. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein n is 0 or 1.-   54. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein n is 0.-   55. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein n is 1.-   56. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein AA2 is γGlu,    αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp, α-D-Asp, or an amino    acid of the following formula:

wherein the arrows indicate the attachment point to the amino group ofAA1, AA2, AA3 or to the ∈-amino group of the B29 lysine residue or to aN-terminal position of the protease stabilised insulin

-   57. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein AA2 is γGlu,    βAsp, γ-D-Glu, β-D-Asp, or an amino acid of the following formula:

wherein the arrow indicate the attachment point to the amino group ofAA1, AA2, AA3 or to the ∈-amino group of the B29 lysine residue or to aN-terminal position of the protease stabilised insulin

-   58. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein AA2 is γGlu,    γ-D-Glu, or an amino acid of the following formula:

wherein the arrow indicate the attachment point to the amino group ofAA1, AA2, AA3 or to the ∈-amino group of the B29 lysine residue or to aN-terminal position of the protease stabilised insulin

-   59. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein m is 0, 1, 2,    3, 4, 5, or 6.-   60. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein m is 0, 1, 2,    3, or 4.-   61. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein m is 4.-   62. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein m is 3.-   63. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein m is 2.-   64. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein m is 1.-   65. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein m is 0.-   66. An acylated protease stabilised insulin, according to any one of    the preceding clauses to the extent possible, wherein AA3 is    selected from any of the following:

wherein r is 1, 2, 3, 5, 7, 11, 23 or 27.

-   67. An acylated protease stabilised insulin, according to the    preceding clause, wherein r is 1, 3, 5, or 7.-   68. An acylated protease stabilised insulin, according to the    preceding clause, wherein r is 1.-   69. An acylated protease stabilised insulin, according to the    preceding clause but one, wherein r is 3.-   70. An acylated protease stabilised insulin, according to the    preceding clause but two, wherein r is 5.-   71. An acylated protease stabilised insulin, according to the    preceding clause but three, wherein r is 7.-   72. An acylated protease stabilised insulin according to any one of    the preceding clauses to the extent possible wherein p is 0, 1, 2,    3, 4, 5, 6, 7, 8, 9, or 10-   73. An acylated protease stabilised insulin according to any one of    the preceding clauses wherein p is 0, 1, 2, 3 or 4.-   74. An acylated protease stabilised insulin according to any one of    the preceding clauses wherein p is 0, 1 or 2.-   75. An acylated protease stabilised insulin according to any one of    the preceding clauses wherein p is 0 or 2.-   76. An acylated protease stabilised insulin according to any one of    the preceding clauses wherein p is 0.-   77. An acylated protease stabilised insulin according to any one of    the preceding clauses wherein p is 1.-   78. An acylated protease stabilised insulin according to any one of    the preceding clauses wherein p is 2.-   79. A compound according to any one of the preceding product    clauses, which is any one of the compounds mentioned specifically in    this specification such as in the specific examples, especially any    one of the examples 1 et seq. below-   80. A compound according to any one of the preceding product    clauses, which is any one of the specific examples of the acyl    moieties mentioned specifically in this specification attached to    any of the protease stabilised insulins mentioned specifically in    this specification.-   81. The use of a compound according to any one of the preceding    product clauses for the preparation of a pharmaceutical composition    for the treatment of diabetes.-   82. The use of a compound according to any one of the preceding    product clauses for the preparation of a pharmaceutical composition    which can be administered pulmonary for the treatment of diabetes.-   83. The use of a compound according to any one of the preceding    product clauses for the preparation of a pharmaceutical composition    which can be administered pulmonary for the treatment of diabetes    and which gives a long acting effect.-   84. The use of a compound according to any one of the preceding    product clauses for the preparation of a powder pharmaceutical    composition which can be administered pulmonary for the treatment of    diabetes.-   85. The use of a compound according to any one of the preceding    product clauses for the preparation of a liquid pharmaceutical    composition which can be administered pulmonary for the treatment of    diabetes.-   86. The use of a compound according to any one of the preceding    product clauses for the preparation of a pharmaceutical composition    which can be administered orally for the treatment of diabetes.-   87. A method of treatment of diabetes, the method comprising    administering to a subject in need thereof a therapeutically    effective amount of a compound according to any one of the preceding    product clauses.-   88. A composition containing human insulin as well as an acylated    protease stabilised insulin according to any one of the preceding    clauses.-   89. A composition containing insulin aspart as well as an acylated    protease stabilised insulin according to any one of the preceding    clauses.-   90. A composition containing insulin Lispro as well as an acylated    protease stabilised insulin according to any one of the preceding    clauses.-   91. A composition containing insulin Glulisine as well as an    acylated protease stabilised insulin according to any one of the    preceding clauses.-   92. A pharmaceutical composition comprising a biologically active    amount of the protease stabilised insulin according to any one of    the above clauses relating to insulin analogs and a pharmaceutically    acceptable carrier.-   93. A method for the treatment, prevention or alleviation of    hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1    diabetes, obesity, syndrome X or dyslipidemia in a subject    comprising administering to a subject an protease stabilised insulin    according to any one of the above clauses relating to insulin    analogs or a pharmaceutical composition according to any one of the    above clauses.-   94. Use of a therapeutically effective amount of an protease    stabilised insulin according to any one of the above clauses    relating to insulin analogs for the preparation of a pharmaceutical    formulation for the treatment or prevention of hyperglycemia, type 2    diabetes, impaired glucose tolerance, type 1 diabetes, obesity,    syndrome X or dyslipidemia.-   95. A method of treatment of diabetes, the method comprising    administering to a subject in need thereof a therapeutically    effective amount of an acylated insulin according to any one of the    preceding product clauses.

Combining one or more of the clauses described herein, optionally alsowith one or more of the claims below, results in further clauses and thepresent invention relates to all possible combinations of said clausesand claims.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents. Thementioning herein of references is no admission that they constituteprior art.

Herein, the word “comprise” is to be interpreted broadly meaning“include”, “contain” or “comprehend” (EPO guidelines C 4.13).

This invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw.

EXAMPLES

The following examples are offered by way of illustration, not bylimitation.

The abbreviations used herein are the following: βAla is beta-alanyl,Aoc is 8-aminooctanoic acid, tBu is tert-butyl, DCM is dichloromethane,DIC is diisopropylcarbodiimide, DIPEA=DIEA is N,N-disopropylethylamine,DMF is N,N-dmethylformamide, DMSO is dimethyl sulphoxide, EtOAc is ethylacetate, Fmoc is 9-fluorenylmethyloxycarbonyl, γGlu is gamma L-glutamyl,HCl is hydrochloric acid, HOBt is 1-hydroxybenzotriazole, NMP isN-methylpyrrolidone, MeCN is acetonitrile, OEG is[2-(2-aminoethoxy)ethoxy]ethylcarbonyl, Su issuccinimidyl-1-yl=2,5-dioxo-pyrrolidin-1-yl, OSu issuccinimidyl-1-yloxy=2,5-dioxo-pyrrolidin-1-yloxy, RPC is reverse phasechromatography, RT is room temperature, TFA is trifluoroacetic acid, THFis tetrahydrofuran, TNBS is 2,4,6-trinitrobenzenesulfonic acid, TRIS istris(hydroxymethyl)aminomethane and TSTU isO—(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate.

The following examples and general procedures refer to intermediatecompounds and final products identified in the specification and in thesynthesis schemes. The preparation of the compounds of the presentinvention is described in detail using the following examples, but thechemical reactions described are disclosed in terms of their generalapplicability to the preparation of compounds of the invention.Occasionally, the reaction may not be applicable as described to eachcompound included within the disclosed scope of the invention. Thecompounds for which this occurs will be readily recognised by thoseskilled in the art. In these cases the reactions can be successfullyperformed by conventional modifications known to those skilled in theart, that is, by appropriate protection of interfering groups, bychanging to other conventional reagents, or by routine modification ofreaction conditions. Alternatively, other reactions disclosed herein orotherwise conventional will be applicable to the preparation of thecorresponding compounds of the invention. In all preparative methods,all starting materials are known or may easily be prepared from knownstarting materials. All temperatures are set forth in degrees Celsiusand unless otherwise indicated, all parts and percentages are by weightwhen referring to yields and all parts are by volume when referring tosolvents and eluents.

The compounds of the invention can be purified by employing one or moreof the following procedures which are typical within the art. Theseprocedures can—if needed—be modified with regard to gradients, pH,salts, concentrations, flow, columns and so forth. Depending on factorssuch as impurity profile, solubility of the insulins in questionetcetera, these modifications can readily be recognised and made by aperson skilled in the art.

After acidic HPLC or desalting, the compounds are isolated bylyophilisation of the pure fractions. After neutral HPLC or anionexchange chromatography, the compounds are desalted, precipitated atisoelectrical pH, or purified by acidic HPLC.

Typical Purification Procedures:

The HPLC system is a Gilson system consisting of the following: Model215 Liquid handler, Model 322-H2 Pump and a Model 155 UV Dector.Detection is typically at 210 nm and 280 nm. The Akta Purifier FPLCsystem (Amersham Biosciences) consists of the following: Model P-900Pump, Model UV-900 UV detector, Model pH/C-900 pH and conductivitydetector, Model Frac-950 Frction collector. UV detection is typically at214 nm, 254 nm and 276 nm.

Acidic HPLC: Column: Macherey-Nagel SP 250/21 Nucleusil 300-7 C4

Flow: 8 ml/minBuffer A: 0.1% TFA in acetonitrileBuffer B: 0.1% TFA in water.

Gradient: 0.0-5.0 min: 10% A 5.00-30.0 min: 10% A to 90% A 30.0-35.0min: 90% A 35.0-40.0 min: 100% A Neutral HPLC: Column: Phenomenex,Jupiter, C4 5 μm 250×10.00 mm, 300 Å

Flow: 6 ml/minBuffer A: 5 mM TRIS, 7.5 mM (NH₄)₂SO₄, pH=7.3, 20% CH₃CNBuffer B: 60% CH3CN, 40% water

Gradient: 0-5 min:10% B 5-35 min: 10-60% B 35-39 min: 60% B 39-40 min:70% B 40-43.5 min: 70% B Anion Exchange Chromatography: Column:RessourceQ, 1 ml

Flow: 6 ml/minBuffer A: 0.09% NH₄HCO₃, 0.25% NH₄OAc, 42.5% ethanol pH 8.4Buffer B: 0.09% NH₄HCO₃, 2.5% NH₄OAc, 42.5% ethanol pH 8.4Gradient: 100% A to 100% B during 30 column volumes

Desalting: Column: HiPrep 26/10

Flow: 10 ml/min, 6 column volumes

Buffer: 10 mM NH₄HCO₃ General Procedure for the Solid Phase Synthesis ofAcylation Reagents of the General Formula (II):

Acy-AA1_(n)-AA2_(m)-AA3_(p)-Act,  (II)

wherein Acy, AA1, AA2, AA3, n, m, and p are as defined above and Act isthe leaving group of an active ester, such as N-hydroxysuccinimide(OSu), or 1-hydroxybenzotriazole, andwherein carboxylic acids within the Acy and AA2 moieties of the acylmoiety are protected as tert-butyl esters.

Compounds of the general formula (II) according to the invention can besynthesised on solid support using procedures well known to skilledpersons in the art of solid phase peptide synthesis. This procedurecomprises attachment of a Fmoc protected amino acid to a polystyrene2-chlorotritylchloride resin. The attachment can, e.g., be accomplishedusing the free N-protected amino acid in the presence of a tertiaryamine, like triethyl amine or N,N-diisopropylethylamine (see referencesbelow). The C-terminal end (which is attached to the resin) of thisamino acid is at the end of the synthetic sequence being coupled to theparent insulins of the invention. After attachment of the Fmoc aminoacid to the resin, the Fmoc group is deprotected using, e.g., secondaryamines, like piperidine or diethyl amine, followed by coupling ofanother (or the same) Fmoc protected amino acid and deprotection. Thesynthetic sequence is terminated by coupling of mono-tert-butylprotected fatty (α, ω) diacids, like hexadecanedioic, heptadecanedioic,octadecanedioic or eicosanedioic acid mono-tert-butyl esters. Cleavageof the compounds from the resin is accomplished using diluted acid like0.5-5% TFA/DCM (trifluoroacetic acid in dichloromethane), acetic acid(e.g., 10% in DCM, or HOAc/trifluoroethanol/DCM 1:1:8), orhecafluoroisopropanol in DCM (See, e.g., “Organic Synthesis on SolidPhase”, F. Z. Dörwald, Wiley-VCH, 2000. ISBN 3-527-29950-5, “Peptides:Chemistry and Biology”, N. Sewald & H.-D. Jakubke, Wiley-VCH, 2002, ISBN3-527-30405-3 or “The Combinatorial Cheemistry Catalog” 1999,Novabiochem AG, and references cited therein). This ensures thattert-butyl esters present in the compounds as carboxylic acid protectinggroups are not deprotected. Finally, the C-terminal carboxy group(liberated from the resin) is activated, e.g., as theN-hydroxysuccinimide ester (OSu) and used either directly or afterpurification as coupling reagent in attachment to parent insulins of theinvention. This procedure is illustrated in example 9.

Alternatively, the acylation reagents of the general formula (II) abovecan be prepared by solution phase synthesis as described below.

Mono-tert-butyl protected fatty diacids, such as hexadecanedioic,heptadecanedioic, octadecanedioic or eicosanedioic acid mono-tert-butylesters are activated, e.g., as OSu-esters as described below or as anyother activated ester known to those skilled in the art, such as HOBt-or HOAt-esters. This active ester is coupled with one of the amino acidsAA1, mono-tert-butyl protected AA2, or AA3 in a suitable solvent such asTHF, DMF, NMP (or a solvent mixture) in the presence of a suitable base,such as DIPEA or triethylamine. The intermediate is isolated, e.g., byextractive procedures or by chromatographic procedures. The resultingintermediate is again subjected to activation (as described above) andto coupling with one of the amino acids AA1, mono-tert-butyl protectedAA2, or AA3 as described above. This procedure is repeated until thedesired protected intermediate Acy-AA1_(n)-AA2_(m)-AA3_(p)-OH isobtained. This is in turn activated to afford the acylation reagents ofthe general formula (II) Acy-AA1_(n)-AA2_(m)-AA3_(p)-Act. This procedureis illustrated in example 21.

The acylation reagents prepared by any of the above methods can be(tert-butyl) de-protected after activation as OSu esters. This can bedone by TFA treatment of the OSu-activated tert-butyl protectedacylation reagent. After acylation of any protease stabilised insulin,the resulting unprotected acylated protease stabilised insulin of theinvention is obtained. This is illustrated eg. in example 16 below.

If the reagents prepared by any of the above methods are not(tert-butyl) de-protected after activation as OSu esters, acylation ofany protease stabilised insulin affords the corresponding tert-butylprotected acylated protease stabilised insulin of the invention. Inorder to obtain the unprotected acylated protease stabilised insulin ofthe invention, the protected insulin is to be de-protected. This can bedone by TFA treatment to afford the unprotected acylated proteasestabilised insulin of the invention. This is illustrated, e.g., inexamples 1 and 2 below.

If acylation of a lysine residue (in the epsilon position) of an insulinis desired, acylation is performed at alkaline pH (eg. at pH 10, 10.5,or 11). This is, e.g., illustrated in examples 1 and 2 below.

If acylation of the A-chain N-terminal position (A1) of an insulin isdesired, acylation is performed at neutral pH (eg. at pH 7, 7.5, 8, or8.5). This is, e.g., illustrated in examples 38, and 44 below.

General Procedure (A) for Preparation of Acylated, Protease StabilisedInsulins of this Invention

The general procedure (A) is illustrated in the first example.

Example 1 General Procedure (A)

A14E, B25H, B29K(N^(∈)-Hexadecandioyl), desB30 Human Insulin

A14E, B25H, desB30 human insulin (500 mg) was dissolved in 100 mMaqueous Na₂CO₃ (5 mL), and pH adjusted to 10.5 with 1 N NaOH.Hexadecanedioic acid tert-butyl ester N-hydroxysuccinimide ester wasdissolved in acetonitrile (10 W/V %) and added to the insulin solutionand heated gently under warm tap, to avoid precipitation and left atroom temperature for 30 minutes. The mixture was lyophilised. The solidwas dissolved in ice-cold 95% trifluoroacetic acid (containing 5% water)and kept on ice for 30 minutes. The mixture was concentrated in vacuoand re-evaporated from dichloromethane. The residue was dissolved inwater, and pH was adjusted to neutral (6-7) and the mixture waslyophilised.

The resulting insulin was purified by ion exchange chromatography on aSource 15Q 21 ml column, several runs, eluting with a gradient of 15 to300 mM ammonium acetate in 15 mM Tris, 50 v/v % ethanol, pH 7.5 (aceticacid). Final desalting of pure fractions were performed on a RPC 3 mLcolumn eluting isocraticlly with 0.1 v/v % TFA, 50 v/v % ethanol. Theresulting pure insulin was lyophilised.

LC-MS (electrospray): m/z=1483.2 (M+4)/4. Calcd: 1483.5

Example 2 General Procedure (A)

A14E, B25H, B29K(N^(∈)Octadecandioyl-γGlu), desB30 Human Insulin

A14E, B25H, desB30 human insulin (2 g) was dissolved in 100 mM aqueousNa₂CO₃ (10 mL), and DMSO (4 mL) was added. pH was adjusted to 10.5 with1 N NaOH. tert-Butyl octadecanedioyl-L-Glu(OSu)-OtBu (prepared asdescribed in WO 2005/012347). More 100 mM aqueous Na₂CO₃ (20 mL) wasadded followed by THF (20 mL). After 1.5 h was a few drops methylamineadded and the mixture was subsequently acidified with acetic acid. Themixture was purified by preparative HPLC and lyophilised to afford thetitle insulin as di-tert-butyl ester. This was dissolved indichloromethane and trifluoroacetic acid 1:1 (50 mL). The mixture wasleft for 2 hours and concentrated in vacuo. After addition of a littlewater and acetonitrile, the mixture was purified by preparative HPLC.Pure fractions were lyophilised. This afforded 313 mg of the titleinsulin.

MALDI-TOF MS: m/z=6089 (M+1). Calcd: 6089.

Example 3 General Procedure (A)

A14E, B25H, B29K(N^(∈)Eicosanedioyl-γGlu), desB30 Human Insulin

This insulin was prepared similarly as described above starting formeicosanedioic acid via eicosanedioic acid mono-tert-butyl ester andtert-butyl icosanedioyl-L-Glu(OSu)-OtBu.

MALDI-TOF MS: m/z=6120 (M+1). Calcd: 6117.

Example 4 General Procedure (A)

A14E, B25H, B29K(N^(∈)3-Carboxy-5-octadecanedioylaminobenzoyl), desB30Human Insulin

This insulin was prepared similarly as described above starting from5-(17-tert-butoxycarbonylhepta-decanoylamino)isophthalic acidmono-(2,5-dioxopyrrolidin-1-yl) ester (prepared as described in WO2006/082204).

LC-MS: 1531 (M+4), Mw 6124 (deconvoluted). Calc.: 1531 (M+4), 6122.

Example 5 General Procedure (A)

A14E, B25H, B29K(N^(∈)—N-octadecandioyl-N-(2-carboxyethyl)glycyl),desB30 Human Insulin

This insulin was prepared similarly as described above starting fromtert-butyl octadecandioyl-N-(2-(tert-butoxycarbonyl)ethyl)-Gly-OSu(prepared as described in WO 2005/012347).

LC-MS (electrospray): m/z: 1522.52 (M+4). Calcd.: 1523.

Example 6 General Procedure (A)

A14E, B25H, B29K(N^(∈)(N-Octadecandioyl-N-carboxymethyl)-beta-alanyl),desB30 Human Insulin

This insulin was prepared similarly as described above starting fromtert-butyl octadecandioyl-N-(tert-butoxycarbonylmethyl)-βAla-OSu(prepared as described in WO 2005/012347).

MALDI-TOF MS: m/z=6088 (M+1). Calcd: 6089.

Example 7 General Procedure (A)

A14E, B25H,B29K(N^(∈)4-([4-({19-Carboxynonadecanoylamino}methyl)trans-cyclohexanecarbonyl]-γGlu),desB30 Human Insulin

This insulin was prepared similarly as described above starting from2-({4-[(19-tert-butoxycarbonyl-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester

LC-MS (electrospray): m/z: 6260. Calcd.: 6255.

Preparation of2-({4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarbonyl}amino)pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl) ester 1. OSuActivation of tea-Butyl Eicosanedioic Acid

tert-Butyl icosanedioic acid (5.0 g) was dissolved in THF (50 ml) andDMF (30 ml). TSTU (4.53 g) and DIPEA (2.65 ml) were added. The mixturewas stirred for 3 days and then concentrated in vacuo. The solid residuewas recrystallized from acetonitrile to give icosanedioic acidtert-butyl ester N-hydroxysuccinimide ester as a white crystallinecompound (5.52 g, 89%).

LC-MS (electrospray): m/z: 440 [M−56 (=tert-Bu)]

2. Coupling of Tranexamic Acid

To a solution of icosanedioic acid tert-butyl ester N-hydroxysuccinimideester (5.52 g) in THF (100 ml) was added tranexamic acid (1.75 g). Aprecipitate was obtained. Attempts to get a solution by adding DMF (75ml), water (25 ml) and DMSO (50 ml) and a few drops of DIPEA were notsuccessful. The suspension was stirred over night. The mixture wasconcentrated in vacuo. To the solid residue was added THF and theprecipitate was filtered off. The filtrate was concentrated and thesolid residue was recrystallized in acetonitrile to give4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarboxylicacid as a white crystalline compound (5.56 g, 93%)

LC-MS (electrospray): m/z: 538 (M+1).

3. OSu Activation of4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarboxylicacid

To a solution of4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarboxylicacid (5.56 g) in THF (100 ml) was added a solution of TSTU (3.42 g) inacetonitrile (25 ml). The mixture was concentrated in vacuo afterstirring over night. The solid residue was recrystallized fromacetonitrite to give4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarboxylicacid 2,5-dioxopyrrolidin-1-yl ester (5.76 g, 88%).

LC-MS (electrospray): m/z: 635 (M+1).

4. Coupling of H-Glu-OtBu and OSu Activation

To a solution of4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarboxylicacid 2,5-dioxopyrrolidin-1-yl ester in THF (150 ml) was added a solutionof H-Glu-OtBu (1.84 g) in water (25 ml) and a few drops of DIPEA. Themixture was stirred over night and then concentrated in vacuo. Theresidue was dissolved in hot (60° C.) THF and filtered. To the coldfiltrate was added THF up to 150 ml and TSTU (2.98 g) dissolved inacetonitrile (25 ml) was added. The mixture was concentrated afterstirring for 20 min. The residue was recrystallized from acetonitrile togive an white solid,2-({4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarbonyl}amino)pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester (6.8 g, 92%).

LC-MS (electrospray): m/z: 820 (M+1).

Example 8 General procedure (A)

A14E. B25H. B29K(N^(∈)Heptadecanedioyl-γGlu). desB30 Human Insulin

This insulin was prepared similarly as described above starting formheptadecanedioic acid via heptadecanedioic acid mono-tert-butyl esterand tert-butyl heptdecanedioyl-L-Glu(OSu)-OtBu (prepared as described inWO 2006/082204).

LC-MS (electrospray): m/z: 1519 (M+4). Calcd.: 1519.

Example 9 General Procedure (A)

A14E, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

The oral effect of this compound on overnight fasted male Wistar rats isgiven in FIG. 2 a and FIG. 2 b below.

This insulin was prepared similarly as described above starting form17-((S)-1-tert-butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)heptadecanoicacid tert-butyl ester (alternative name: tert-Butyloctadecandioyl-Glu(OEG-OEG-OSu)-OtBU)

LC-MS (electrospray): m/z: 1596 (M+4). Calcd.: 1596.

The building block for preparation of this insulin was prepared asdescribed in the following:

Starting resin: 2-Chlorotrityl resin, 1.60 mmol/g

1.0 g of the resin was swelled for 30 min in DCM (10 ml).

1. Acylation with Fmoc-8-amino-3,6-dioxaoctanoic acid

0.39 g (0.63 eq, 1.0 mmol) of Fmoc-8-amino-3,6-dioxaoctanoic acid(Fmoc-OEG-OH) was dissolved in DCM (15 ml) and was added to the resin.N,N-Diisopropylethylamine (DIEA) (0.44 ml, 2.5 mmol) was added dropwise.The reaction mixture was vortexed for 30 min. and then methanol (2 ml)was added and the mixture was vortexed for additional 15 min. The resinwas filtered and washed with NMP (2×8 ml) and DCM (8×8 ml).

20% piperidine/NMP (8 ml) was added, standing 10 min. repeated once.Filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml). Apositive TNBS test gave red-coloured resins.

2. Acylation with Fmoc-8-amino-3,6-dioxaoctanoic acid

0.78 g (2 eq, 2.0 mmol) of Fmoc-8-amino-3,6-dioxaoctanoic acid wasdissolved in NMP/DCM 1:1 (10 ml). 0.28 g (2.2 eq, 2.4 mmol) of HOSu wasadded followed by addition of 0.37 ml (2.2 eq, 2.4 mmol) of DIC. Thereaction mixture was allowed to stand for 1 hour and was then added tothe resin and finally 0.407 ml (2.2 eq) of DIEA was added. The mixturewas vortexed for 16 hours, filtered and washed with NMP (2×8 ml), DCM(3×8 ml), and NMP (5×8 ml). A positive TNBS test gave colourless resins.

20% piperidine/NMP (10 ml) was added, standing 10 min. repeated once.Filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml). Apositive TNBS test gave red-coloured resins.

Acylation with Fmoc-Glu-OtBu:

0.86 g (2 eq, 2.0 mmol) of Fmoc-Glu-OtBu was dissolved in NMP/DCM 1:1(10 ml). 0.32 g (2.2 eq, 2.4 mmol) of HOBT was added followed byaddition of 0.37 ml (2.2 eq, 2.4 mmol) of DIC. The reaction mixture wasallowed to stand for 20 min and was then transferred to the resin andfinally 0.407 ml (2.2 eq) of DIEA was added. The mixture was vortexedfor 16 hours, filtered and washed with NMP (2×8 ml), DCM (3×8 ml), andNMP (5×8 ml). A positive TNBS test gave colourless resins.

20% piperidine/NMP (10 ml) was added, standing 10 min. repeated once.Filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml). Apositive TNBS test gave red-coloured resins.

Acylation with Octadecanedioic Acid Mono Tert-Butyl Ester:

0.75 g (2 eq, 2.0 mmol) Octadecanedioic acid mono tert-butyl ester wasdissolved NMP/DCM 1:1 (10 ml). 0.32 g (2.2 eq, 2.4 mmol) HOBT was addedfollowed by addition of 0.37 ml (2.2 eq, 2.4 mmol) of DIC. The reactionmixture was allowed to stand for 20 min and was then transferred to theresin and finally 0.41 ml (2.2 eq) of DIEA was added. The mixture wasvortexed for 16 hours, filtered and washed with NMP (2×8 ml), DCM (3×8ml), and NMP (5×8 ml).

Cleavage with TFA:

8 ml of 5% TFA/DCM was added to the resin and the reaction mixture wasvortexed for 2 hours, filtered and the filtrate was collected. More 5%TFA/DCM (8 ml) was added to the resin, and the mixture was vortexed for10 min, filtered and the resin was washed with DCM (2×10 ml). Thecombined filtrates and washings were pH adjusted to basic using about800 ul of DIEA. The mixture was evaporated in vacuo affording an oil(3.5 g). Diethylether (30 ml) was added and the not dissolved oil wasseparated by decantation and evaporated in vacuo. This afforded 1.1 g of17-{(S)-1-tert-butoxycarbonyl-3-[2-(2-{[2-(2-carboxymethoxyethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}heptadecanoicacid tert-butyl ester (alternative name: tert-butyloctadecandioyl-Glu(OEG-OEG-OH)-OTBU) as an oil.

LC-MS (Sciex100 API): m/z=846.6 (M+1)⁺.

OSu-Activation:

The above tert-butyl octadecandioyl-Glu(OEG-OEG-OH)-OtBU (0.63 g) wasdissolved in THF (35 ml). DIEA (0.255 ml, 2 eq.) was added followed byTSTU (0.45 g, 2 eq.), and the mixture was stirred at room temperaturefor 16 hours. The mixture was partitioned between ethyl acetate (250 ml)and aqueous NaHSO4 (3×100 ml). The organic phase was dried (MgSO4) andconcentrated in vacuo to afford 0.65 g of17-((S)-1-tert-butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)heptadecanoicacid tert-butyl ester (alternative name: tert-butyloctadecandioyl-Glu(OEG-OEG-OSu)-OtBu) as an oil.

LC-MS: m/z=943.4 (M+1).

Example 10 General Procedure (A)

A14E, B25H, B29K(N^(∈)Myristyl), desB30 Human Insulin

This insulin was prepared similarly as described above starting form1-tetradecanoyl-pyrrolidine-2,5-dione.

MALDI-TOF MS: m/z=5873.6. Calcd: 5872.9.

Example 11 General Procedure (A)

A14E, B25H, B29K(N^(∈)Eicosanedioyl-γGlu-γGlu), desB30 Human Insulin

This insulin was prepared similarly as described above starting from(S)-2-[4-tert-butoxycarbonyl-4-(19-tert-butoxycarbonylnonadecanoylamino)butyrylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester.

MALDI-TOF MS: m/z=6242.5. Calcd: 6245.2.

Preparation of(S)-2-[4-tert-butoxycarbonyl-4-(19-tert-butoxycarbonylnonadecanoylamino)butyrylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester 1.(S)-2-[4-tert-Butoxycarbonyl-4-(19-tert-butoxycarbonylnonadecanoylamino)butyrylamino]-pentanedioicacid 1-tert-butyl ester

To a solution of(S)-2-(19-tert-butoxycarbonylnonadecanoylamino)pentanedioic acid1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester (prepared similarlyas described in WO 2005/012347) (4.1 g) in THF (100 ml) was added asolution of H-Glu-OtBu (1.47 g) in water (20 ml). pH was adjusted to 8with DIPEA. The mixture was concentrated after stirring for 1.5 h. Theresidue was recrystallized from DCM to give the title compound as awhite solid (2.81 g, 61%).

LC-MS: m/z=769 (M+1).

(S)-2-[4-tert-Butoxycarbonyl-4-(19-tert-butoxycarbonylnonadecanoylamino)butyrylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester

To a solution of(S)-2-[4-tert-butoxycarbonyl-4-(19-tert-butoxycarbonylnonadecanoylamino)butyrylamino]pentanedioicacid 1-tert-butyl ester (2.81 g) in acetonitrile (80 ml) was added asolution of TSTU (1.32 g) in acetonitrile (20 ml). pH was adjusted to 8with DIPEA. After stirring for 1.5 h the mixture was concentrated. Theresidue was recrystallized from acetonitrile to give the title compound(1.7 g, 54%).

LC-MS: m/z=866.4 (M+1).

Example 12 General Procedure (A)

A14E, B25H,B29K(W4-([4-({19-Carboxynonadecanoylamino}methyl)trans-cyclohexanecarbonyl]-γGlu-γGlu),desB30 Human Insulin

This insulin was prepared similarly as described above starting from2-[4-tert-butoxycarbonyl-4-({4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]-pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester

LC-MS (electrospray): m/z: 6386 (M+1). Calcd.: 6384.

Preparation of2-[4-tert-butoxycarbonyl-4-({4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]-cyclohexanecarbonyl}amino)butyrylamino]pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester 1.2-[4-tert-Butoxycarbonyl-4-({4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]pentanedioicacid 1-tert-butyl ester

To a solution of2-({4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarbonyl}-amino)pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester (5.0 g) in THF(100 ml) was added a solution of H-Glu-OtBu (1.36 g) in water (25 ml).After stirring over night the mixture was concentrated in vacuo. Theresidue was precipitated from water and filtered off and dried in vacuoto give the title compound (4.63 g, 84%).

LC-MS: m/z=740 (M−3×56, loss of 3xt-Bu).

2-[4-tert-Butoxycarbonyl-4-({4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester

To a solution of2-[4-tert-butoxycarbonyl-4-({4-[(19-tert-butoxycarbonylnonadecanoylamino)methyl]-cyclohexanecarbonyl}amino)butyrylamino]pentanedioicacid 1-tert-butyl ester (4.6 g) in THF (150 ml) was added TSTU (1.68 g).DIPEA (0.97 ml) was added. After stirring over night the mixture wasconcentrated in vacuo. The residue was crystallized from acetonitrile toafford the title compound as a solid (4.4 g, 87%)

LC-MS: m/z=837 (M−3×56, loss of 3xt-Bu).

Example 13 General Procedure (A)

A14E, B25H, B29K (N^(∈)Octadecanedioyl-γGlu-γGlu), desB30 Human Insulin

The oral effect of this compound on overnight fasted male Wistar rats isgiven in FIG. 7 below.

LC-MS: m/z=1555 (M+4)14.

Example 14 General Procedure (A)

A14E, B28D, B29K(N^(∈) octadecandioyl-γGlu), desB30 Human Insulin

Example 15 General Procedure (A)

A14E, B25H, B29K(N^(∈) octadecandioyl-γGlu-PEG7), desB30 Human Insulin

MALDI-TOF MS: m/z=6510

Example 16 General Procedure (A)

A14E, B25H, B29K(N^(∈)eicosanedioyl-γGlu-OEG-OEG), desB30 human Insulin

The oral effect of this compound on overnight fasted male Wistar rats isgiven in FIG. 3 below.

MALDI-TOF MS: m/z=6407

The intermediate acylation reagent for this example was prepared asdescribed in the following:

Step 1:19-{(S)-1-tert-Butoxycarbonyl-3-[2-(2-{[2-(2-carboxymethoxy-ethoxy)-ethylcarbamoyl]-methoxy}-ethoxy)-ethylcarbamoyl]-propylcarbamoyl}-nonadecanoicacid tert-butyl ester

To a solution of 2-(19-tert-Butoxycarbonylnonadecanoylamino)pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester (2.50 g,(prepared similarly as described in WO 2005/012347) and[2-(2-{2-[2-(2-Aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetic acid(1.47 g, alternative name: 8-amino-3,6-dioxaoctanoic acid dimer, IRISBiotech GmbH, Cat. No. PEG1221) in ethanol (40 ml) was added DIPEA (1.26ml). The mixture was stirred at room temperature over night and thenconcentrated in vacuo. To the residue was added aqueous 0.1 N HCl (150ml) and ethyl acetate (200 ml). The layers were separated and theaqueous layer was extracted with ethyl acetate (100 ml). The combinedorganic layeres were washed with water and brine, dried (magnesiumsulphate) and concentrated in vacuo to give an oil, which crystalised onstanding. Yield 96% (3.1 g). LC-MS (electrospray): m/z=874.49.

Step 2:19-((S)-1-tert-Butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoicacid tert-butyl ester

To a solution of19-{(S)-1-tert-Butoxycarbonyl-3-[2-{[2-(2-carboxymethoxyethoxy)ethylcarbamoyl]-methoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}nonadecanoicacid tert-butyl ester (3.1 g) in acetonitrile (50 ml) was added TSTU(1.39 g) and DIPEA (0.91 ml). The mixture was stirred at roomtemperature over night and then concentrated in vacuo. To the residuewas added aqueous 0.1 N HCl (100 ml) and ethyl acetate (200 ml). Thelayers were separated and the aqueous layer was extracted with ethylacetate (50 ml). The combined organic layers were washed with water andbrine, dried (magnesium sulphate) and concentrated in vacuo to give anoil. Yield 99% (3.4 g). LC-MS (electrospray): m/z: 971.8.

Step 3:19-((S)-1-Carboxy-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoicacid

19-((S)-1-tert-Butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoicacid tert-butyl ester (3.4 g) was stirred in TFA (75 ml) for 45 min andthen concentrated in vacuo. The residue was concentrated with toluene 3times to give a solid. The residue was crystallised in 2-propanol andfiltered to give a white crystalline compound. Yield 80% (2.4 g). LC-MS(electrospray): m/z: 859.44.

The similar acylation reagent with the octadecanedioic acid fragment (egused in example 26 and other examples) can be prepared similarly.

Example 17 General Procedure (A)

A14E, B25H,B29K(N^(∈)eicosanedioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu),desB30 Human Insulin

ES-MS: m/z=1626 (M+4)

Example 18 General Procedure (A)

A14E, B25H, B29K(N^(∈)Hexadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

MALDI-TOF MS: m/z=6348

Example 19 General Procedure (A)

A14E, B25H, B29K(N^(∈)Hexadecanedioyl-γGlu), desB30 Human Insulin

MALDI-TOF MS: m/z=6062

Example 20 General Procedure (A)

A14E, B25H, B29K(N^(∈)heptadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

ES-MS: m/z=1592 (M+4)

Example 21 General Procedure (A)

A14E, B25H, B29K(N^(∈)octadecanedioyl-γGlu-γGlu-γGlu-γGlu), desB30 HumanInsulin

ES-MS: m/z=1620 (M+4)

The intermediate acylation reagentoctadecanedioyl-γGlu-γGlu-γGlu-γGlu-OSu (with tert-butyl esters asprotection groups on remaining carboxylic acids) was prepared asdescribed below:

Octadecanedioic acid tert-butyl ester 2,5-dioxopyrrolidin-1-yl ester

Octadecanedioic acid mono-tert-butyl ester (4.2 g, 0.011 mol) wasdissolved in THF (20 mL), TSTU (4 g, 0.013 mol) in acetonitrile (20 mL)was added and pH of the solution was adjusted to 8 with dropwiseaddition of DIPEA. The mixture was stirred at RT for 4 h, then acidifiedwith HCl (2M) to pH 3 and evaporated in vacuo. The residual oil wassubsequently partitioned between ethyl acetate and HCl (0.1 M). Theorganic layer was dried (MgSO₄), filtered and evaporated to dryness invacuo. This afforded 5.2 g of octadecanedioic acid tert-butyl ester2,5-dioxopyrrolidin-1-yl ester as an oil, which could be used in thenext step without further purification. LC-MS (electrospray): m/z=468(M+1) and 412 (M+1-^(t)Bu).

(S)-2-(17-tert-Butoxarbonylheptadecanoylamino)-pentanedioic acid1-tert-butyl ester

Octadecanedioic acid tert-butyl ester 2,5-dioxopyrrolidin-1-yl ester (7g, 0.015 mol) was dissolved in THF (80 mL) and added to a solution ofH-Glu-O^(t)Bu (3.7 g, 0.0165 mol) in Na₂CO₃ (0.1 M, 40 mL). The mixturewas stirred at RT overnight, then acidified with HCl (2M) to pH 3 andevaporated in vacuo. The residue was partitioned between ethyl acetateand HCl (0.1 M). The organic layer was dried (MgSO₄), filtered andevaporated to dryness in vacuo. Addition of acetonitrile (30 mL) causedthe formation of a white precipitate, which was isolated by filtrationto and dried to afford 3.75 g of(S)-2-(17-tert-butoxarbonylheptadecanoylamino)pentanedioic acid1-tert-butyl ester. LC-MS (electrospray): m/z=556 (M+1).

On evaporation of the acetonitrile filtrate further 2.6 g of product wasisolated.

(S)-2-(17-tert-Butoxycarbonylheptadecanoylamino)pentanedioic acid1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl) ester

(S)-2-(17-tert-Butoxarbonylheptadecanoylamino)pentanedioic acid1-tert-butyl ester (3 g, 0.005 mol) was dissolved in THF (100 mL) andadded to a solution of TSTU (1.78 g, 0.006 mol) in acetonitrile (30 mL).pH was adjusted to 8 by dropwise addition of DIPEA. The mixture wasstirred at RT for 1 h, then acidified with HCl (2M) to pH 3 andevaporated in vacuo. The residual oil was subsequently partitionedbetween ethyl acetate and HCl (0.1 M). The organic layer was dried(MgSO₄), filtered and evaporated in vacuo to dryness. This afforded awhite solid (2.75 g) of(S)-2-(17-tert-butoxycarbonylheptadecanoylamino)-pentanedioic acid1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester. LCMS(electrospray): m/z=653 (M+1).

(S)-2-[(S)-4-tert-Butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]-pentanedioicacid 1-tert-butyl ester

(S)-2-(17-tert-Butoxycarbonylheptadecanoylamino)pentanedioic acid1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester (0.5 g, 0.766 mmol)was dissolved in acetonitrile (20 mL). This solution was added to asolution of H-Glu-OtBu (0.171 g, 0.84 mmol) in water (30 mL) pH wasadjusted to 10 with DIPEA. The mixture was stirred at RT for 15 min,then acidified to pH 7 with HCl (2M) and evaporated in vacuo. Theresidue was partitioned between ethyl acetate and HCl (0.1 M). Theorganic layer was dried (MgSO₄), filtered, and evaporated in vacuo todryness. This afforded(S)-2-[(S)-4-tert-butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]pentanedioicacid 1-tert-butyl ester as an oil. LC-MS (electrospray): m/z=741 (M+1).

(S)-2-[(S)-4-tert-Butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]-pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester

(S)-2-[(S)-4-tert-Butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]-pentanedioicacid 1-tert-butyl ester (8 g, 10.79 mmol) was dissolved in acetonitrile(40 mL) and a solution of TSTU (3.89 g, 12.95 mmol) in acetonitrile (40mL) was added. pH was adjusted to 8 by dropwise addition of DIPEA. Themixture was stirred at RT for 1 h, then acidified with HCl (2M) to pH 3and evaporated in vacuo. This afforded an oil, which was subsequentlypartitioned between ethyl acetate and HCl (0.1 M). The organic layer wasdried (MgSO₄), filtered and evaporated to dryness in vacuo. Thisafforded 8.2 g of(S)-2-[(S)-4-tert-butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester as a solid.

(S)-2-{(S)-4-tert-Butoxycarbonyl-4-[(S)-4-tert-butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]butyrylamino}pentanedioicacid 1-tert-butyl ester

(S)-2-[(S)-4-tert-Butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]-pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester (4 g, 4.77mmol) was dissolved in acetonitrile (30 mL) and added to a solution ofH-Glu-OtBu (1.07 g, 5.25 mmol) in Na₂CO₃ (0.1 M, 20 mL). The mixture wasstirred at RT for 1 h, then neutralised with HCl (2M) to pH 7 andevaporated in vacuo. The residual oil was subsequently partitionedbetween ethyl acetate and HCl (0.1 M). The organic layer was dried(MgSO₄), filtered and evaporated to dryness in vacuo. The residue (4 g)was dissolved in acetonitrile and treated with active carbon. Afterfilteration and evaporation to dryness followed by drying overnight invacuo, 2.8 g of(S)-2-{(S)-4-tert-Butoxycarbonyl-4-[(S)-4-tert-butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]butyrylamino}pentanedioicacid 1-tert-butyl ester was obtained as a crystalline solid. LC-MS(electrospray): m/z=927 (M+1).

(S)-2-((S)-4-tert-Butoxycarbonyl-4-{(S)-4-tert-butoxycarbonyl-4-[(S)-4-tert-butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butylamino]butyrylamino}butyrylamino)-pentanedioicacid 1-tert-butyl ester

(S)-2-{(S)-4-tert-Butoxycarbonyl-4-[(S)-4-tert-butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]butyrylamino}pentanedioicacid 1-tert-butyl ester (2.8 g, 3.02 mmol) was activated with TSTU (1.0g, 3.325 mmol) using the same method as described above, giving crude(S)-2-{(S)-4-tert-butoxycarbonyl-4-[(S)-4-tert-butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]butyrylamino}-pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester. LCMS(electrospray): m/z=1024 (M+1).

1.3 g of this compound was dissolved in acetonitrile (40 mL) and addedto a solution of H-Glu-O^(t)Bu (0.28 g, 1.39 mmol) in water (30 mL), pHwas adjusted to 9.3 with DIPEA. The mixture was stirred at RT for 2 h,then neutralised to pH 7 with HCl (2M) and then evaporated in vacuo toalmost dryness. The residue was treated with water giving a whiteprecipitate, which was filtered off. After drying in vacuo overnight,1.1 g of(S)-2-((S)-4-tert-butoxycarbonyl-4-{(S)-4-tert-butoxycarbonyl-4-[(S)-4-tertbutoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]butyrylamino}butyrylamino)pentanedioicacid 1-tert-butyl ester was isolated, containing minor amounts ofstarting material.

LC-MS (electrospray): m/z=1111.9 (M+1).

(S)-2-((S)-4-tert-Butoxycarbonyl-4-{(S)-4-tert-butoxycarbonyl-4-[(S)-4-tert-butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]butyrylamino}butyrylamino)-pentanedioicacid 1-tert-butylester-5-(2,5-dioxopyrrolidin-1-yl)ester

(S)-2-((S)-4-tert-Butoxycarbonyl-4-{(S)-4-tert-butoxycarbonyl-4-[(S)-4-tert-butoxycarbonyl-4-(17-tert-butoxycarbonylheptadecanoylamino)butyrylamino]butyrylamino}butyrylamino)pentanedioicacid 1-tert-butyl ester (0.1 g, 0.09 mmol) was activated with TSTU (29.8mg, 0.099 mmol) in acetonitrile solution at RT for 1 h using the samemethod for activation and work up as described above. This afforded 100mg crude activated product which could be used as such for insulinacylation without further purification. LC-MS (electrospray): m/z=1208(M+1).

Example 22 General Procedure (A)

A14E, B25H, B29K(N^(∈)Eicosanedioyl-γGlu-γGlu-γGlu), desB30 HumanInsulin

MALDI-TOF MS: m/z=6373

Example 23 General Procedure (A)

A14E, B25H, B27E, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

MALDI-TOF MS: m/z=6407

Example 24 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG),desB30 Human Insulin

The oral effect of this compound on overnight fasted male Wistar rats isgiven in FIG. 6 below.

MALDI-TOF MS: m/z=6188

Example 25 General Procedure (A)

A14E, B16H, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

The oral effect of this compound on overnight fasted male Wistar rats isgiven in FIG. 4 below.

MALDI-TOF MS: m/z=6352

Example 26 General Procedure (A)

A14E, B16E, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 humaninsulin

MALDI-TOF MS: m/z=6345

Example 27 General Procedure (A)

A14E, B16H, B25H, B29K(N^(∈)Hexadecanedioyl-γGlu), desB30 Human Insulin

The oral effect of this compound on overnight fasted male Wistar rats isgiven in FIG. 5 below.

MALDI-TOF MS: m/z=6041

Example 28 General Procedure (A)

A14E, B25H, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-γGlu), desB30 Human Insulin

ES-MS: m/z=1598 (M+4)

Example 29 General Procedure (A)

A14E, B16E, B25H, B29K(N^(∈)Hexadecandioyl-γGlu), desB30 Human Insulin

MALDI-TOF MS: m/z=6028

Example 30 General Procedure (A)

A14E, B16H, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-γGlu-γGlu), desB30Human Insulin

ES-MS: m/z=1581 (M+4)

Example 31 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Hexadecandioyl-γGlu), desB30Human Insulin

ES-MS: m/z=1484 (M+4)

Example 32 General Procedure (A)

A14E, B16H, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-γlu), desB30 HumanInsulin

ES-MS: m/z=1548 (M+4)

Example 33 General Procedure (A)

A14E, B16H, B25H, B29K(N(eps)Eicosanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

ES-MS: m/z=1596 (M+4)

Example 34 General Procedure (A)

A14E, B25H, B29K(N^(∈)Octadecanedioyl-OEG-γGlu-γGlu), desB30 HumanInsulin

ES-MS: m/z=1592 (M+4)

Example 35 General Procedure (A)

A14E, A18L, B25H, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

MALDI-TOF MS: m/z=6405

Example 36 General Procedure (A)

A14E, A18L, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

MALDI-TOF MS: m/z=6377

Example 37 General Procedure (A)

A14E, B25H, B27E, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-OEG), desB30 Humaninsulin

MALDI-TOF MS: m/z=6433

Example 38 General Procedure (A)

A1G(N^(α)Octadecandioyl-γGlu-OEG-OEG), A14E, B25H, B29R, desB30 humaninsulin

A14E, B25H, B29R, desB30 insulin (500 mg, 88 μmol) was dissolved in 0.1M NaHCO₃, pH 8 (5 mL). w-carboxyheptadecanoyl-γ-L-glutamyl-OEG-OEG-OSu(65 mg, 88 μmol) was dissolved in THF/MeCN 1:1 (5 mL) and added to theinsulin solution. After 30 minutes, the reaction was quenched byaddition of 2 M aqueous methylamine (0.5 mL). The solvent was evaporatedin vacuo and the solid was redissolved in the minimal amount ofwater/MeCN. The main product peak was isolated by use of RP-HPLC on C18column, buffer A: 0.1% TFA in water, buffer B: 0.1% TFA in MeCN,gradient 30-55% buffer B over 45 mins. The product fractions werepartially evaporated in vacuo and freeze-dried to provide 59 mg product(10%). LC-MS analysis: M⁴⁺=1602.7, calculated 1602.6. Two steps ofstandard amino acid sequence analysis showed F-V, confirming theacylation at A1.

Example 39 General Procedure (A)

A14E, B1F(N^(α)Octadecandioyl-γGlu-OEG-OEG), B25H, B29R, desB30 HumanInsulin

This compound was isolated as a byproduct from the example above(example 38). LCMS analysis: M⁴⁺=1602.5, calculated 1602.6. Two steps ofstandard amino acid sequence analysis showed G-I, confirming theacylation at B1.

Example 40 General Procedure (A)

A1G(N^(α)Hexadecandioyl-γGlu), A14E, B25H, B29R, desB30 Human Insulin

ES-MS: m/z=1523 (M+4)

This compound was prepared similarly to the A1-acylation described above(example 38), using ω-carboxypentadecanoyl-γ-L-glutamyl(OSu) asacylation reagent. The product showed LCMS: M⁴⁺=1523.2, calculated1523.0. Two steps of standard amino acid sequence analysis showed F-V,confirming the acylation at A1.

Example 41 General Procedure (A)

A14E, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-Abu-Abu-Abu-Abu), desB30Human Insulin

ES-MS: m/z=1286 (M+5)

The acylation reagent for this example was prepared in analogy with thereagent prepared in example 9, starting with attachment of Fmocprotected 4-aminobutyric acid to 2-chlorotrityl resin, followed bydeprotection and sequential attachment 3 more units of 3 Fmoc protected4-aminobutyric acid, and as described in example 9, Fmoc-Glu-OtBu andoctadecanedioic acid mono-tert-butyl ester.

Example 42 General Procedure (A)

A14E, B25H, B29K(N^(α)Eicosanedioyl), desB30 Human Insulin

MALDI-TOF-MS: m/z=5987

Example 43 General Procedure (A)

A14E, B25H,B29K(N^(α)4-[16-(1H-Tetrazol-5-yl)hexadecanoylsulfamoyl]butanoyl),desB30 Human Insulin

ES-MS: m/z=1530 (M+4)

Preparation of the Intermediateacylation Reagent:

4-[16-(1H-Tetrazol-5-yl)hexadecanoylsulfamoyl]butanoic acid (500 mg,prepared as described in WO 2006/005667) was dissolved in ethanol (20ml), and TSTU (381 mg), and DIPEA (542 μl) were added and the resultingmixture was stirred at room temperature for 16 hours. The mixture wasconcentrated in vacuo, and the residue was stirred with 0.25M HCl. Thesolid was isolated by filtration, washed with water and dried in vacuoto afford 580 mg (91%) of the acylation reagent.

Acylation Reaction:

A14E, B25H, desB30 human insulin (500 mg) was dissolved in 0.1 M aqueoussodium carbonate (10 mL) and ethanol (4 mL). pH was adjusted to 10.8with 1N NaOH. The above acylation reagent (101 mg) dissolved in THF (2mL) and ethanol (2 mL) was added in two portions with 10 minutesinterval.

The resulting mixture was stirred slowly for 1 hour and diluted withwater (50 mL). The resulting insulin was precipitated by addition of 1NHCl to pH 5.5. The precipitate was isolated by centrifugation andpurified by HPLC. Pure fractions were pooled and lyophilised.

Example 44 General Procedure (A)

A1G(N^(α)Octadecandioyl-γGlu-OEG-OEG), A14E, A21G, B25H, desB30 HumanInsulin

MALDI-TOF-MS: m/z=6321

Example 45 General Procedure (A)

A14E, B25H, B29K(N^(∈)Eicosanedioyl-OEG), desB30 Human Insulin

MALDI-TOF-MS: m/z=6130

Example 46 General Procedure (A)

A14E, B25H, B27K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB28, desB29,desB30 Human Insulin

MALDI-TOF-MS: m/z=6181

Example 47 General Procedure (A)

A14E, B25H, B29K(N^(∈)(5-Eicosanedioylaminoisophthalic acid)), desB30Human Insulin

MALDI-TOF-MS: m/z=6150

Example 48 General Procedure (A)

A14E, B25H, B29K(N^(∈)Octadecanedioyl), desB30 Human Insulin

MALDI-TOF-MS: m/z=5959

Example 49 General Procedure (A)

A14E, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 Human Insulin

ES-MS: m/z=1598 (M+4)

Example 50 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-OEG),desB30 Human Insulin

MALDI-TOF-MS: m/z=6216

Example 51 General Procedure (A)

A14E, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-OEG), desB30 Human Insulin

ES-MS: m/z=1559 (M+4)

Example 52 General Procedure (A)

A14E, B25H, B29K(N^(∈)Eicosanedioyl-OEG-OEG), desB30 Human Insulin

MALDI-TOF-MS: m/z=6278

Example 53 General Procedure (A)

A14E, B25H, B29K(N^(∈)Eicosanedioyl-Aoc), desB30 Human Insulin

MALDI-TOF-MS: m/z=6126

Example 54 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Eicosanedioyl-γGlu-γGlu), desB30Human Insulin

ES-MS: m/z=6055 (deconvoluted)

Example 55 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Eicosanedioyl-γGlu-γGlu), desB30Human Insulin

ES-MS: m/z=6220 (deconvoluted)

Example 56 General Procedure (A)

A14E, B25H, B29K(N^(∈)Octadecanedioyl-OEG), desB30 Human Insulin

MALDI-TOF-MS: m/z=6101

Example 57 General Procedure (A)

A14E, B25H, desB27, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30Human Insulin

MALDI-TOF-MS: m/z=6277

Example 58 General Procedure (A)

A14E, B25H, B16H, B29K(N^(∈)Octadecanedioyl-γGlu), desB30 Human Insulin

ES-MS: m/z=1516 (M+4)

Example 59 General Procedure (A)

A1G(N^(α)Octadecanedioyl), A14E, B25H, B29R, desB30 Human Insulin

ES-MS: m/z=1498 (M+4)

Example 60 General Procedure (A)

A14E, B16H, B25H, B29K(N^(∈)Eicosanedioyl-γGlu), desB30 Human Insulin

ES-MS: m/z=1523 (M+4)

Example 61 General Procedure (A)

A14E, B25H, B27K(N^(∈)Eicosanedioyl-γGlu), desB28, desB29, desB30 HumanInsulin

MALDI-TOF MS: m/z=6208

Example 62 General Procedure (A)

A14E, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-γGlu-γGlu), desB30 HumanInsulin

ES-MS: m/z=1587 (M+4)

The acylated insulins of the invention in following examples may beprepared similarly:

Example 63 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Octadecandioyl-γGlu), desB30Human Insulin

Example 64 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Eicosanedioyl-γGlu), desB30Human Insulin

Example 65 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Octadecandioyl), desB30 HumanInsulin

Example 66 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Eicosanedioyl), desB30 HumanInsulin

Example 67 General Procedure (A)

A14E, B25H, B29K(N^(∈)Docosanedioyl-γGlu), desB30 Human Insulin

Example 68 General Procedure (A)

A14E, B25H, B29K(N^(∈)Docosanedioyl-γGlu-γGlu), desB30 Human Insulin

Example 69 General Procedure (A)

A14E, B25H, B29K(N^(∈)Icosanedioyl-γGlu-OEG-OEG-γGlu), desB30 HumanInsulin

Example 70 General Procedure (A)

A14E, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG-γGlu), desB30 HumanInsulin

Example 71 General Procedure (A)

A14E, B25H, B29K(N^(∈)(N-Icosanedioyl-N-carboxymethyl)-βAla), desB30Human Insulin

Example 72 General Procedure (A)

A14E, B25H,B29K(N^(∈)3-[2-(2-{2-[2-(17-Carboxyheptadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-γGlu),desB30 Human Insulin

Example 73 General Procedure (A)

A14E, B25H,B29K(N^(∈)3-[2-(2-{2-[2-(19-Carboxynonadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-γGlu),desB30 Human Insulin

Example 74 General Procedure (A)

A14E, B25H,B29K(N^(∈)Octadecandioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl),desB30 Human Insulin

Example 75 General Procedure (A)

A14E, B25H,B29K(N^(∈)Octadecandioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu),desB30 Human Insulin

Example 76 General Procedure (A)

A14E, B25H,B29K(N^(∈)Icosanedioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl),desB30 Human Insulin

Example 77 General Procedure (A)

A14E, B25H,B29K(N^(∈)4-([4-({17-Carboxynonadecanoylamino}methyl)trans-cyclohexanecarbonyl]-γGlu),desB30 Human Insulin

Example 78 General Procedure (A)

A14E, B25H,B29K(N^(∈)4-([4-({17-Carboxyheptadecanoylamino}methyl)trans-cyclohexanecarbonyl]-γGlu-γGlu),desB30 Human Insulin

Example 79 General Procedure (A)

A14E, B28D, B29K(N^(∈) hexadecandioyl-γGlu), desB30 Human Insulin

Example 80 General Procedure (A)

A14E, B28D, B29K(N^(∈) Eicosanedioyl-γGlu), desB30 Human Insulin

Example 81 General Procedure (A)

A14E, B28D, B29K(N^(∈) Octadecandioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 82 General Procedure (A)

A14E, B28D, B29K(N^(∈) Eicosanedioyl-γGlu-OEG-OEG), desB30 Human Insulin

Example 83 General Procedure (A)

A14E, B28E, B29K(N^(∈) Hexadecandioyl-γGlu), desB30 Human Insulin

Example 84 General Procedure (A)

A14E, B28E, B29K(N^(∈) Octadecandioyl-γGlu), desB30 Human Insulin

Example 85 General Procedure (A)

A14E, B28E, B29K(N^(∈) Eicosanedioyl-γGlu), desB30 Human Insulin

Example 86 General Procedure (A)

A14E, B28E, B29K(N^(∈) Octadecandioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 87 General Procedure (A)

A14E, B28E, B29K(N^(∈) Eicosanedioyl-γGlu-OEG-OEG), desB30 Human Insulin

Example 88 General Procedure (A)

A14E, B1E, B28E, B29K(N^(∈) Hexadecandioyl-γGlu), desB30 Human Insulin

Example 89 General Procedure (A)

A14E, B1E, B28E, B29K(N^(∈) Octadecandioyl-γGlu), desB30 Human Insulin

Example 90 General Procedure (A)

A14E, B1E, B28E, B29K(N^(∈) Eicosanedioyl-γGlu), desB30 Human Insulin

Example 91 General Procedure (A)

A14E, B1E, B28E, B29K(N^(∈) Hexadecandioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 92 General Procedure (A)

A14E, B1E, B28E, B29K(N^(∈) Octadecandioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 93 General Procedure (A)

A14E, B1E, B28E, B29K(N^(∈) Eicosanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 94 General Procedure (A)

A14E, B1E, B27E, B28E, B29K(N^(∈) Hexadecandioyl-γGlu), desB30 HumanInsulin

Example 95 General Procedure (A)

A14E, B1E, B27E, B28E, B29K(N^(∈) Octadecandioyl-γGlu), desB30 HumanInsulin

Example 96 General Procedure (A)

A14E, B1E, B27E, B28E, B29K(N^(∈) Eicosanedioyl-γGlu), desB30 HumanInsulin

Example 97 General Procedure (A)

A14E, B1E, B27E, B28E, B29K(N^(∈) Hexadecandioyl-γGlu-OEG-OEG), desB30Human Insulin

Example 98 General Procedure (A)

A14E, B1E, B27E, B28E, B29K(N^(∈) Octadecandioyl-γGlu-OEG-OEG), desB30Human Insulin

Example 99 General Procedure (A)

A14E, B1E, B27E, B28E, B29K(N^(∈) Eicosanedioyl-γGlu-OEG-OEG), desB30Human Insulin

Example 100 General Procedure (A)

A14E, B1E, B25H, B28E, B29K(N^(∈) Hexadecandioyl-γGlu), desB30 HumanInsulin

Example 101 General Procedure (A)

A14E, B1E, B25H, B28E, B29K(N^(∈) Octadecandioyl-γGlu), desB30 HumanInsulin

Example 102 General Procedure (A)

A14E, B1E, B25H, B28E, B29K(N^(∈) Eicosanedioyl-γGlu), desB30 HumanInsulin

Example 103 General Procedure (A)

A14E, B1E, B25H, B28E, B29K(N^(∈) Hexadecandioyl-γGlu-OEG-OEG), desB30Human Insulin

Example 104 General Procedure (A)

A14E, B1E, B25H, B28E, B29K(N^(∈) Octadecandioyl-γGlu-OEG-OEG), desB30Human Insulin

Example 105 General Procedure (A)

A14E, B1E, B25H, B28E, B29K(N^(∈) Eicosanedioyl-γGlu-OEG-OEG), desB30Human Insulin

Example 106 General Procedure (A)

A14E, B1E, B25H, B27E, B28E, B29K(N^(∈) Hexadecandioyl-γGlu), desB30Human Insulin

Example 107 General Procedure (A)

A14E, B1E, B25H, B27E, B28E, B29K(N^(∈) Octadecandioyl-γGlu), desB30Human Insulin

Example 108 General Procedure (A)

A14E, B1E, B25H, B27E, B28E, B29K(N^(∈) Eicosanedioyl-γGlu), desB30Human Insulin

Example 109 General Procedure (A)

A14E, B1E, B25H, B27E, B28E, B29K(N^(∈) Hexadecandioyl-γGlu-OEG-OEG),desB30 Human Insulin

Example 110 General Procedure (A)

A14E, B1E, B25H, B27E, B28E, B29K(N^(∈)Octadecandioyl-γGlu-OEG-OEG),desB30 Human Insulin

Example 111 General Procedure (A)

A14E, B1E, B25H, B27E, B28E, B29K(N^(∈) Eicosanedioyl-γGlu-OEG-OEG),desB30 Human Insulin

Example 112 General Procedure (A)

A14E, B28D, B29K(N^(∈)Hexadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 113 General Procedure (A)

A14E, B28E, B29K(N^(∈)Hexadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 114 General Procedure (A)

B25N, B27E, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-OEG), desB30 Human Insulin

Example 115 General Procedure (A)

B25N, B27E, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 116 General Procedure (A)

B25N, B27E, B29K(N^(∈)Hexadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 117 General Procedure (A)

B25N, B27E, B29K(N^(∈)Eicosanedioyl-γGlu), desB30 Human Insulin

Example 118 General Procedure (A)

B25N, B27E, B29K(N^(∈)Octadecanedioyl-γGlu), desB30 Human Insulin

Example 119 General Procedure (A)

B25N, B27E, B29K(N^(∈)Hexadecanedioyl-γGlu), desB30 Human Insulin

Example 120 General Procedure (A)

A8H, B25N, B27E, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 121 General Procedure (A)

A8H, B25N, B27E, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 122 General Procedure (A)

A8H, B25N, B27E, B29K(N^(∈)Hexadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 123 General Procedure (A)

A8H, B25N, B27E, B29K(N^(∈)Eicosanedioyl-γGlu), desB30 Human Insulin

Example 124 General Procedure (A)

A8H, B25N, B27E, B29K(N^(∈)Octadecanedioyl-γGlu), desB30 Human Insulin

Example 125 General Procedure (A)

A8H, B25N, B27E, B29K(N^(∈)Hexadecanedioyl-γGlu), desB30 Human Insulin

Example 126 General Procedure (A)

A14E, B25H, B29K(N^(∈)(N-Icosanedioyl-N-carboxymethyl)-βAla-OEG-OEG),desB30 Human Insulin

Example 127 General Procedure (A)

A14E, B25H, B29K(N^(∈)(N-Octadecanedioyl-N-carboxymethyl)-βAla-OEG-OEG),desB30 Human Insulin

Example 128 General Procedure (A)

A14E, B25H, B29K(N^(∈)(N-Hexadecanedioyl-N-carboxymethyl)-βAla-OEG-OEG),desB30 Human Insulin

Example 129 General Procedure (A)

A14E, B25H,B29K(N^(∈)octadecanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]acetyl),desB30 Human Insulin

[(3-{2-[2-(3-Aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]aceticacid may prepared as described (Eur. J. Med. Chem. 2007, 42, 114) andreacted with ω-(tert-butyl-carboxy-heptadecanoyl-γ-L-glutamyl(OSu)-OtBu.The product may be activated using TSTU and coupled to A14E, B25H,desB30 human insulin in 0.1 M Na₂CO₃ at pH 10.5 to provide the product.

Example 130 General Procedure (A)

A14E, B25H,B29K(N^(∈)eicosanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]acetyl),desB30 Human Insulin

[(3-{2-[2-(3-Aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]aceticacid may be prepared as described (Eur. J. Med. Chem. 2007, 42, 114) andreacted with w-(tert-butyl-carboxy-nonadecanoyl-γ-L-glutamyl(OSu)-OtBu.The product may be activated using TSTU and coupled to A14E, B25H,desB30 human insulin in 0.1 M Na₂CO₃ at pH 10.5 to provide the product.

Example 131 General Procedure (A)

A14E, B16H, B25H,B29K(N^(∈)Octadecanediovl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]acetyl),desB30 Human Insulin

[(3-{2-[2-(3-Aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]aceticacid may prepared as described (Eur. J. Med. Chem. 2007, 42, 114) andreacted with ω-(tert-butyl-carboxy-heptadecanoyl-γ-L-glutamyl(OSu)-OtBu.The product may be activated using TSTU and coupled to A14E, B16H, B25H,desB30 human insulin in 0.1 M Na₂CO₃ at pH 10.5 to provide the product.

Example 132 General Procedure (A)

A14E, B16H, B25H,B29K(N^(∈)Eicosanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]acetyl),desB30 Human Insulin

[(3-{2-[2-(3-Aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]aceticacid may be prepared as described (Eur. J. Med. Chem. 2007, 42, 114) andreacted with ω-(tert-butyl-carboxy-nonadecanoyl-γ-L-glutamyl(OSu)-OtBu.The product may be activated using TSTU and coupled to A14E, B16H, B25H,desB30 human insulin in 0.1 M Na₂CO₃ at pH 10.5 to provide the product.

Example 133 General Procedure (A)

B25H, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 Human Insulin

Example 134 General Procedure (A)

B25H, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-OEG), desB30 Human Insulin

Example 135 General Procedure (A)

B25H, B29K(N^(∈)Octadecanedioyl-γGlu), desB30 Human Insulin

Example 136 General Procedure (A)

B25H, B29K(N^(∈)Eicosanedioyl-γGlu), desB30 Human Insulin

Example 137 General Procedure (A)

B25H, B29K(N^(∈)Octadecanedioyl), desB30 Human Insulin

Example 138 General Procedure (A)

B25H, B29K(N^(∈)Eicosanedioyl), desB30 Human Insulin

Example 139 General Procedure (A)

B25H, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 Human Insulin

Example 140 General Procedure (A)

B25H, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-OEG), desB30 Human Insulin

Example 141 General Procedure (A)

B25H, B29K(N^(∈)Octadecanedioyl-γGlu), desB30 Human Insulin

Example 142 General Procedure (A)

B25H, B29K(N^(∈)Eicosanedioyl-γGlu), desB30 Human Insulin

Example 143 General Procedure (A)

A21G, B25H, B29K(N^(∈)Octadecanedioyl), desB30 Human Insulin

Example 144 General Procedure (A)

A21G, B25H, B29K(N^(∈)Eicosanedioyl), desB30 Human Insulin

Example 145 General Procedure (A)

A21G, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 146 General Procedure (A)

A21G, B25H, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-OEG), desB30 Human Insulin

Example 147 General Procedure (A)

A21G, B25H, B29K(N^(∈)Octadecanedioyl-γGlu), desB30 Human Insulin

Example 148 General Procedure (A)

A21G, B25H, B29K(N^(∈)Eicosanedioyl-γGlu), desB30 Human Insulin

Example 149 General Procedure (A)

A14E, B25H, desB27, B29K(N^(∈)Octadecanedioyl), desB30 Human Insulin

Example 150 General Procedure (A)

A14E, B25H, desB27, B29K(N^(∈)Eicosanedioyl), desB30 Human Insulin

Example 151 General Procedure (A)

A14E, B25H, desB27, B29K(N^(∈)Octadecanedioyl-γGlu), desB30 HumanInsulin

Example 152 General Procedure (A)

A14E, B25H, desB27, B29K(N^(∈)Eicosanedioyl-γGlu), desB30 Human Insulin

Example 153 General Procedure (A)

A14E, B25H, desB27, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 154 General Procedure (A)

A14E, A21G, B25H, desB27, B29K(N^(∈)Octadecanedioyl), desB30 HumanInsulin

Example 155 General Procedure (A)

A14E, A21G, B25H, desB27, B29K(N^(∈)Eicosanedioyl), desB30 Human Insulin

Example 156 General Procedure (A)

A14E, A21G, B25H, desB27, B29K(N^(∈)Octadecanedioyl-γGlu), desB30 HumanInsulin

Example 157 General Procedure (A)

A14E, B25H, desB27, B29K(N^(∈)Eicosanedioyl-γGlu), desB30 Human Insulin

Example 158 General Procedure (A)

A14E, A21G, B25H, desB27, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG),desB30 Human Insulin

Example 159 General Procedure (A)

A14E, A21G, B25H, desB27, B29K(N^(∈)Eicosanedioyl-γGlu-OEG-OEG), desB30Human Insulin

Example 160 General Procedure (A)

A14E, A21G, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 161 General Procedure (A)

A14E, A21G, B25H, B29K(N^(∈) Eicosanedioyl-γGlu-OEG-OEG), desB30 HumanInsulin

Example 162 General Procedure (A)

A14E, A21G, B25H, B29K(N^(∈)Eicosanedioyl-γGlu), desB30 Human Insulin

Example 163 General Procedure (A)

A14E, A21G, B25H, B29K(N^(∈)Eicosanedioyl), desB30 Human Insulin

Example 164 General Procedure (A)

A14E, A21G, B25H, B29K(N^(∈) Octadecanedioyl-γGlu), desB30 Human Insulin

Example 165 General Procedure (A)

A14E, A21G, B25H, B29K(N^(∈) Octadecanedioyl), desB30 Human Insulin

Example 166 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈) Octadecanedioyl-γGlu), desB30Human Insulin

Example 167 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Octadecanedioyl), desB30 HumanInsulin

Example 168 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Eicosanedioyl-γGlu), desB30Human Insulin

Example 169 General Procedure (A)

A14E, B25H, B26G, B27G, B28G, B29K(N^(∈)Eicosanedioyl), desB30 HumanInsulin

Example 170 General Procedure (A)

A1G(N^(α)Octadecandioyl-γGlu), A14E, B25H, B26G, B27G, B28G, desB30Human Insulin

Example 171 General Procedure (A)

A1G(N^(α)Eicosanedioyl-γGlu), A14E, B25H, B26G, B27G, B28G, desB30 HumanInsulin

Example 172 General Procedure (A)

A1G(N^(α)Octadecandioyl-γGlu), A14E, B25H, B26G, B27G, B28G, B29R,desB30 Human Insulin

Example 173 General Procedure (A)

A1G(N^(α)Eicosanedioyl-γGlu), A14E, B25H, B26G, B27G, B28G, B29R, desB30Human Insulin

Example 174 General Procedure (A)

A1G(N^(α)Octadecandioyl), A14E, B25H, B26G, B27G, B28G, desB30 HumanInsulin

Example 175 General Procedure (A)

A1G(N^(α)Eicosanedioyl), A14E, B25H, B26G, B27G, B28G, desB30 HumanInsulin

Example 176 General Procedure (A)

A1G(N^(α)Octadecandioyl), A14E, B25H, B26G, B27G, B28G, B29R, desB30Human Insulin

Example 177 General Procedure (A)

A1G(N^(α)Eicosanedioyl), A14E, B25H, B26G, B27G, B28G, B29R, desB30Human Insulin

Example 178 Insulin Receptor Affinity of Selected Insulin Derivatives ofthe Invention

The affinity of the acylated insulin analogues of this invention for thehuman insulin receptor is determined by a SPA assay (ScintillationProximity Assay) microtiterplate antibody capture assay. SPAPVTantibody-binding beads, anti-mouse reagent (Amersham Biosciences, CatNo. PRNQ0017) are mixed with 25 ml of binding buffer (100 mM HEPES pH7.8; 100 mM sodium chloride, 10 mM MgSO₄, 0.025% Tween-20). Reagent mixfor a single Packard Optiplate (Packard No. 6005190) is composed of 2.4μl of a 1:5000 diluted purified recombinant human insulin receptor(either with or without exon 11), an amount of a stock solution ofA14Tyr[¹²⁵I]-human insulin corresponding to 5000 cpm per 100 μl ofreagent mix, 12 μl of a 1:1000 dilution of F12 antibody, 3 ml ofSPA-beads and binding buffer to a total of 12 ml. A total of 100 μlreagent mix is then added to each well in the Packard Optiplate and adilution series of the insulin derivative is made in the Optiplate fromappropriate samples. The samples are then incubated for 16 hours whilegently shaken. The phases are the then separated by centrifugation for 1min and the plates counted in a Topcounter. The binding data were fittedusing the nonlinear regression algorithm in the GraphPad Prism 2.01(GraphPad Software, San Diego, Calif.) and affinities are expressedrelative (in pertcentage (%)) to the affinity of human insulin.

A related assay is also used wherein the binding buffer also contains4.5% HSA in order to mimic physiological conditions

Insulin receptor affinities of selected insulins of the invention:

Relative IR-A Relative IR-A affinity affinity Example # (@ 0% HSA) (%)(@ 4.5% HSA) (%) 19 3.8 .30 10 9.5 1 5.0 .10 2 2.1 .06 5 2.5 4 3.4 3 2.09 1.7 .20 6 2.6 .04 7 2.1 8 2.1 12 1.7 11 .8 17 .9 13 1.1 15 1.9 20 2.022 .7 16 .9 .23 18 2.3 23 1.4 24 7.9 2.23 25 .4 .05 26 .0 .01 27 .7 .0628 .3 29 .2 .01 30 .3 .02 31 16.2 1.11 32 .3 33 .5 0.06 21 .8 34 1.3 355.8 36 9.3 37 .8 40 0.3 38 .6 .10 41 1.6 .31 39 11.2 .67 Prior 10 1.00art 183 46 1.9 0.08 47 1.2 0.10 48 1.3 0.01 49 6.2 0.86 50 4.3 1.21 511.7 0.12 52 2.1 53 2.3 0.03 54 3.9 0.91 55 0.3 0.03 56 4.4 0.03 57 2.558 0.5 59 0.3

Example 179 Hydrophobicity of the Insulin Derivatives of the Invention

The hydrophobicity of an insulin derivative is found by reverse phaseHPLC run under isocratic conditions. The elution time of the insulinderivative is compared to that of human insulin (herein designated HI)or another derivative with a known hydrophibicity under the sameconditions. The hydrophobicity, k′rel, is calculated as:k′rel_(deriv)=((t_(deriv)−t₀)/(t_(ref)−t₀)*k′rel_(ref). Using HI asreference: k′rel_(ref)=k′rel_(HI)=1. The void time of the HPLC system,t₀, is determined by injecting 5 μl of 0.1 mM NaNO₃. Running conditions:

Column: Lichrosorb RP-C18, 5 μm, 4×250 mm

Buffer A: 0.1 M natrium phosphate pH 7.3, 10 vol % CH₃CN

Buffer B: 50 vol % CH₃CN

Injection volume: 5 μlRun time: max 60 minutes

After running an initial gradient, the isocratic level for running thederivative and reference (for example HI) is chosen, and the elutiontimes of the derivative and reference under isocratic conditions areused in the above equation to calculate k′rel_(deriv).

Relative hydrophobicity, Example # k'rel_(deriv) 19 .07 10 14.60 1 .33 2.25 5 .23 4 .48 3 .77 9 .31 6 .19 7 2.78 8 .14 12 .94 11 .19 17 .57 13.10 15 .43 20 .15 22 .20 16 1.15 18 .10 23 .16 24 .26 25 .22 26 .21 27.05 28 .42 29 .05 30 .05 31 32 .07 33 .76 21 .04 34 35 .84 36 .24 37 .5640 .09 38 41 46 0.44

Example 180 Pulmonary Delivery of Insulin Derivatives to Rats Protocol:

The test substance will be dosed pulmonary by the drop instillationmethod. In brief, male Wistar rats (app. 250 g) are anaesthesized inapp. 60 ml fentanyl/dehydrodenzperidol/-dormicum given as a 6.6 ml/kg scpriming dose and followed by 3 maintenance doses of 3.3 ml/kg sc with aninterval of 30 min. Ten minutes after the induction of anaesthesia,basal samples are obtained from the tail vein (t=−20 min) followed by abasal sample immediately prior to the dosing of test substance (t=0). Att=0, the test substance is dosed intra tracheally into one lung. Aspecial cannula with rounded ending is mounted on a syringe containingthe 200 ul air and test substance (1 ml/kg). Via the orifice, thecannula is introduced into the trachea and is forwarded into one of themain bronchi—just passing the bifurcature. During the insertion, theneck is palpated from the exterior to assure intratracheal positioning.The content of the syringe is injected followed by 2 sec pause.Thereafter, the cannula is slowly drawn back. The rats are keptanaesthesized during the test (blood samples for up to 4 or 8 hrs) andare euthanized after the experiment.

FIGS. 8 and 9 show blood glucose lowering effects and plasma insulinconcentrations, respectively, from intratracheal drop instillation of aninsulin of the invention (example 9), compared with a similar, butnon-protease resistant insulin of the prior art (example 183).

Example 181 Pulmonary Delivery of Insulin Derivatives to Mini-PigsProtocol:

The pigs were instrumented with central venous catheters for intravenousinjections and blood sampling. The pigs are fasted prior to thepulmonary experiment, i.e. the day before dosing, the leftovers from theafternoon feeding is removed approximately one hour after feeding and onthe day of dosing, the pigs are not fed. The patency of the catheters ischecked prior to the experiment with saline added 10 IU/ml heparin.

After pulmonary dosing, a glucose solution should be ready for i.v.injection to prevent hypoglycaemia, i.e. 4-5 syringes (20 ml) are filledwith sterile 20% glucose, ready for use. Diagnosis of hypoglycemia isbased on clinical symptoms and blood glucose measurements on aglucometer (Glucocard X-meter). Treatment consists of slow i.v.injection 50-100 ml 20% glucose (10-20 g glucose). The glucose is givenin fractions over 5-10 minutes until effect.

The pigs are fasted during the first part of the experiment (until 24h), but with free access to water. After the 16 h blood sample cathetersare closed with 5000 IU/ml heparin, placed in the pockets and the pigsare released. After the 24 h blood sample the pigs are fed with doubleration of food and apples. Pigs are not fasted from 24 h to 48 h.

Compound and Pulmonary Dosing Powder for Pulmonary Dosing

The insulin powders are weighed into 8 separate powder chamber of thedry powder device (PennCentury™ Model DP-4, custom made porcine device)the day before the experiment. All chambers are kept protected fromlight and humidity by keeping them on a desiccating material in acontainer with aluminumfoil around in a temperature and humiditycontrolled laboratory until dosing.

Based on the most recent individual animal weight, the delivery devicewas preloaded with 25 nmol/kg as some powder retention was expected.

Loading dose=(Weight of powder+(weight of device and powder−weight ofdevice))/2.

Anaesthesia

By an i.v. injection of Domitor® Vet inj. (medetomidein 1 mg/ml), 0.15ml/10 kg=0.4 ml/pig, the pig is sedated.

Immediately after, Rapinovet Vet. inj. (propofol 10 mg/ml) is injectedslowly i.v. until sufficient depth of anaesthesia is obtained. Ingeneral, 2-3 ml/10 kg is enough, but it may be necessary to supplementwith 1-2 ml at a time until intubation is possible. Atropin (1 mg/ml) isinjected i.m. at 0.5 ml/pig and allowed to work min. 5 minutes beforeintubation.

For intubation the pig is placed in ventral position with slightlyelevated front, local anaesthetics Xylocalne® kutanspray (lidocain 10mg/dosis) is sprayed onto the epiglottis, and the pigs are intubatedusing a laryngoscope and a disposable tube size 8.0 mm (ID). The twoparts of the tube are pressed tightly together.

Device Position During Pulmonal Dosing

The position of the PennCentury™ device during dosing should be justoutside the end of the endotracheal tube and this should be measured onthe device before intubation (remember the connecting L-piece whenmeasuring this). During dosing the tip of the PennCentury™ device shouldpositioned in the trachea just below the bronchus that goes to lobuscranialis dexter, which is confirmed with the bronchoscope.

Artificial Respiration

The respiration frequency is set to 10/min and the respiration depth to250 ml/breath. The respirator is mounted with “baby” bag to optimisetiming of dosing. The anaesthesia apparatus is connected to a filterthat is connected to the endotracheal tube via a L-piece. ThePennCentury™ device is introduced through the L-piece, which will allowcontrol over the respiration depth and frequency during dosing.

Dosing Technique

The PennCentury™ device should be placed as described above. The pigsare dosed (one at a time) with the PennCentury™ device by manualadministration during inhalation using the adjustable PennCentury airpump (Model AP-1). Each pig is given 8 air sprays (air pump set to 4 mL)during 8 consecutive respirator-forced inhalations to ensure that theentire dose is given. The chamber is gently tapped between sprays toavoid sticking of the powder to the device. A new delivery tube is usedfor each pig. The timing in relation to the inhalation is veryimportant, and the air sprays should be given in the very beginning ofthe inhalation (aim for start at 50 mL inhalation).

To counteract the effect of Domitor, Antisedan® Vet inj. (atipamezol 5mg/ml) will be injected as an intramuscular injection (0.4 ml/pig)immediately after dosing, and the pigs will be taken back to their pensand allowed to wake up from anaesthesia.

Retention Analysis

The emitted dose should be the entire content of the chamber and afterdosing the device is weighed again with any residual powder, and theretained powder is extracted with 9 ml of 0.01 N HCl med 0.05% (w/v)Tween 80 extraction buffer and sent to analysis.

Blood Sampling

After the dosing, blood samples will be taken from a central venouscatheter at the following time points:

−10, 0, 10, 20, 40, 60, 90, 120, 150, 180, 240 (4 h), 300 (5 h), 360 (6h), 8 h, 10 h, 12 h, 14 h, 16 h, 24 h, 32 h and 48 h.

Samples are taken with a 3-way stop-cock; waste blood is injected backinto the animal. Sample size is: 0.8 ml of blood collected in a tubecoated with EDTA. After each blood sample the catheter is flushed with 5ml of sterile 0.9% NaCl with 10 IU/ml heparin. The tube is tilted gentlya minimum of 8 times to ensure sufficient mixing of blood andanticoagulant (EDTA) and after one minute it is placed on wet ice. Thetubes are spun for 10 min at 3000 rpm and 4° C. within 1 hour aftersampling. The samples are stored on wet ice until pipetting.

Closure of the Catheters after the Experiment

A single intravenous treatment with Ampicillin (10 mg/kg=0.1 ml/kg of a100 mg/ml solution) dissolved in sterile saline (1 g Ampicillin in 10ml=100 mg/ml) is given via the catheter that has been used for bloodsampling. Both catheters are flushed with 4-5 ml of sterile 0.9% NaCladded heparin to a concentration to 10 IU/ml. The catheters are closedwith a new luer-lock with latex injection membrane. 4-5 ml sterile 0.9%NaCl is injected through the membrane. Finally 0.8 ml of heparin, 5000IU/ml, is injected through the catheter as a lock. Aseptic technique isdemanded to avoid bacterial growth in the catheter with increased riskof clotting.

Analysis of Blood Samples

10 μl of plasma is pippetted into 500 μl of EBIO buffer solution formeasurements of glucose concentration in plasma in the Biosenautoanalyser.

Plasma samples are also assayed for exogenous insulin by immunoassays tocalculate PK parameters.

Pulmonary Dosing of the Insulin of Example 9 to Mini-Pigs According tothe Protocol Above:

FIGS. 10 and 11 shows the pharmacokinetic profile of the insulin ofexample 9 compared to the same insulin but without the proteasestabilising A14E and B25H mutations (insulin of prior art). The data arefrom the same experiment, FIG. 10 is shown with the data from the first250 minutes, and FIG. 11 is shown with the full 24 hour (1440 minutes)time-course.

Pharmacokinetic data for the insulin of example 9 compared to the sameinsulin but without the protease stabilising A14E and B25H mutations(insulin of prior art). The data are from the same experiment, half-life(T½) and bioavailability (F_(it)) relative to intravenousadministration:

Insulin, T_(1/2) example # (minutes) F_(it) Prior art 211  4% (see ex.183) 9 1127 13%

Example 182 Degradation of Insulin Analogs Using Duodenum Lumen Enzymes

Degradation of insulin analogs using duodenum lumen enzymes (prepared byfiltration of duodenum lumen content) from SPD rats. The assay isperformed by a robot in a 96 well plate (2 ml) with 16 wells availablefor insulin analogs and standards. Insulin analogs ˜15 μM are incubatedwith duodenum enzymes in 100 mM Hepes, pH=7.4 at 37° C., samples aretaken after 1, 15, 30, 60, 120 and 240 min and reaction quenched byaddition of TFA. Intact insulin analogs at each point are determined byRP-HPLC. Degradation half time is determined by exponential fitting ofthe data and normalized to half time determined for the referenceinsulins, A14E, B25H, desB30 human insulin or human insulin in eachassay. The amount of enzymes added for the degradation is such that thehalf time for degradation of the reference insulin is between 60 min and180 min. The result is given as the degradation half time for theinsulin analog in rat duodenum divided by the degradation half time ofthe reference insulin from the same experiment (relative degradationrate).

Duodenum degradation. Relative stability vs. Duodenum degradation. A14E,B25H, desB30 Relative stability vs. Example # human insulin humaninsulin 19 1.8 21.6 2 1.3 15.6 3 .7 8.4 9 .8 9.6 8 1.8 21.6 11 .9 10.813 1.5 18 22 .9 11 16 .5 6 18 1.1 13.2 23 1.9 22.8 24 1.2 14.4 25 1.113.2 26 1.2 14.4 27 2.9 35 28 .7 7.2 29 3.1 37 30 2.1 25.2 31 1.6 19.232 1.9 22.8 33 .5 6 21 1.1 13.2 34 1.0 12 35 .6 7.2 36 .9 10.8 37 .8 9.640 .7 8.4 38 .5 6 41 .7 8.4 Prior 0.1 1.2 art 183 46 2.0 24 47 0.6 7 480.5 6 49 0.1 1.2 50 0.5 6 51 1.0 12

Rat Pharmacokinecics: Intravenous rat PK:

Anaesthetized rats are dosed intravenously (i.v.) with insulin analogsat various doses and plasma concentrations of the employed compounds aremeasured using immunoassays or mass spectrometry at specified intervalsfor 4 hours or more post-dose. Pharmacokinetic parameters aresubsequently calculated using WinNonLin Professional (Pharsight Inc.,Mountain View, Calif., USA).

Non-fasted male Wistar rats (Taconic) weighing approximately 200 gramare used.

Body weight is measured and rats are subsequently anaesthetized withHypnorm/Dormicum (each compound is separately diluted 1:1 in sterilewater and then mixed; prepared freshly on the experimental day).Aanaesthesia is initiated by 2 ml/kg Hypnorm/Doricum mixture sc followedby two maintenance doses of 1 ml/kg sc at 30 min intervals and twomaintenance doses of 1 ml/kg sc with 45 min intervals. If required inorder to keep the rats lightly anaesthetised throughout a furtherdose(s) 1-2 ml/kg sc is supplied. Weighing and initial anaesthesia isperformed in the rat holding room in order to avoid stressing theanimals by moving them from one room to another.

Peroral Rat PK: Gavage:

Conscious rats are p.o. dosed with insulin analogs. Plasmaconcentrations of the employed compounds as well as changes in bloodglucose are measured at specified intervals for 4-6 hours post-dosing.Pharmacokinetic parameters are subsequently calculated using WinNonLinProfessional (Pharsight Inc., Mountain View, Calif., USA)

Male Sprague-Dawley rats (Taconic), weighing 250-300 g are fasted for˜18 h and p.o. dosed with test compound or vehicle.

The Composition of the Formulation Used for the Oral Gavage Dosing isthe Following (in Weight %):

45% Propylene glycol (Merck) 33% Capmul MCM C10 (Abitec) 11% Poloxamer407 (BASF) 11% Polyethyleneglycol 3350 Ultra (Fluka)

The amount of added insulin is subtracted equaly from Capmul MCM C10,Poloxamer 407 and PEG 3350 and not from propylene glycol in order tokeep the amount of propylene glycol independent of the drug loadconstant at 45%.

Neutral insulin (freeze-dried from pH 7.4) is dissolved in propyleneglycol at RT under gentle agitation. Depending on the insulin and theamount of insulin it can take a few hours to dissolve in propyleneglycol. The resulting solution should be clear. The other additives,Capmul, poloxamer and PEG3350 are mixed and melted together at 58 C andshould also result in a clear, slightly yellowish solution. Then theinsulin propylene glycol solution is warmed up to 35° C. and the meltedadditives are added portionwise under magnetic stirring. The resultingmixture should be clear and homogenously at 35° C. and results in a semisolid after storage in the fridge. After preparation the SEDDScomposition is cooled down to 5° C. in order to solidify.

Blood samples for the determination of whole blood glucoseconcentrations are collected in heparinised 10 μl capillary tubes bypuncture of the capillary vessels in the tail tip. Blood glucoseconcentrations are measured after dilution in 500 μl analysis buffer bythe glucose oxidase method using a Biosen autoanalyzer (EKF DiagnosticGmbh, Germany). Mean blood glucose concentration courses (mean±SEM) aremade for each compound.

Samples are collected for determination of the plasma insulinconcentration. 100 μl blood samples are drawn into chilled tubescontaining EDTA. The samples are kept on ice until centrifuged (7000rpm, 4° C., 5 min), plasma is pipetted into Micronic tubes and thenfrozen at 20° C. until assay. Plasma concentrations of the insulinanalogs are measured in the Assay and Technology dept. using animmunoassay which is considered appropriate or validated for theindividual analog.

Blood samples are drawn at t=−10 (for blood glucose only), at t=−1 (justbefore dosing) and at specified intervals for 4-6 hours post-dosing.

Intraintestinal Injection:

Anaesthetized rats are dosed intraintestinally (into jejunum) withinsulin analogs. Plasma concentrations of the employed compounds as wellas changes in blood glucose are measured at specified intervals for 4hours or more post-dosing. Pharmacokinetic parameters are subsequentlycalculated using WinNonLin Professional (Pharsight Inc., Mountain View,Calif., USA).

Male Sprague-Dawley rats (Taconic), weighing 250-300 g, fasted for ˜18 hare anesthetized using Hypnorm-Dormicum s.c. (0.079 mg/ml fentanylcitrate, 2.5 mg/ml fluanisone and 1.25 mg/ml midazolam) 2 ml/kg as apriming dose (to timepoint −60 min prior to test substance dosing), 1ml/kg after 20 min followed by 1 ml/kg every 40 min.

The insulins to be tested in the intraintestinal injection model areformulated as formulated for the gavage model above.

The anesthetized rat is placed on a homeothermic blanket stabilized at37° C. A 20 cm polyethylene catheter mounted a 1-ml syringe is filledwith insulin formulation or vehicle. A 4-5 cm midline incision is madein the abdominal wall. The catheter is gently inserted into mid-jejunum˜50 cm from the caecum by penetration of the intestinal wall. Ifintestinal content is present, the application site is moved ±10 cm. Thecatheter tip is placed approx. 2 cm inside the lumen of the intestinalsegment and fixed without the use of ligatures. The intestines arecarefully replaced in the abdominal cavity and the abdominal wall andskin are closed with autoclips in each layer. At time 0, the rats aredosed via the catheter, 0.4 ml/kg of test compound or vehicle.

Blood samples for the determination of whole blood glucoseconcentrations are collected in heparinised 10 μl capillary tubes bypuncture of the capillary vessels in the tail tip. Blood glucoseconcentrations are measured after dilution in 500 μl analysis buffer bythe glucose oxidase method using a Biosen autoanalyzer (EKF DiagnosticGmbh, Germany). Mean blood glucose concentration courses (mean±SEM) aremade for each compound.

Samples are collected for determination of the plasma insulinconcentration. 100 μl blood samples are drawn into chilled tubescontaining EDTA. The samples are kept on ice until centrifuged (7000rpm, 4° C., 5 min), plasma is pipetted into Micronic tubes and thenfrozen at 20° C. until assay. Plasma concentrations of the insulinanalogs are measured in a immunoassay which is considered appropriate orvalidated for the individual analog.

Blood samples are drawn at t=−10 (for blood glucose only), at t=−1 (justbefore dosing) and at specified intervals for 4 hours or morepost-dosing.

MRT (min) (Mean retention Fpo, Gavage Fpo, Intraintestinal Example #time, gavage) (%) injection (%) 183 (Prior  97 ± 15 0.005 ± 0.009 0.28 ±0.14 art) 2 401 ± 82 0.05 ± 0.02 9  345 ± 111 0.10 ± 0.09 1.9 ± 1.0 13251 ± 47 0.12 ± 0.08 16 416 ± 37 0.06 ± 0.04 1.8 ± 1.9 18 149 ± 29 0.13± 0.07 24 194 ± 54 0.06 ± 0.05 1.4 ± 1.1 25  481 ± 108 0.20 ± 0.05 3.3 ±1.2 26 33

Rat Pharmacodynamics:

Blood glucose vs. time profiles following oral administration (asdescribed above) of selected acylated insulins of the invention areshown below:

Example 183

The oral effect of overnight fasted male Wistar rats on an insulin ofthe prior art, i.e.,:

B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 human insulin is givenin FIG. 1 below.

Example 184 Potency of the Acylated Insulin Analogues of this InventionRelative to Human Insulin

Sprague Dawley male rats weighing 238-383 g on the experimental day areused for the clamp experiment. The rats have free access to feed undercontrolled ambient conditions and are fasted overnight (from 3 μm) priorto the clamp experiment.

Experimental Protocol:

The rats are acclimatized in the animal facilities for at least 1 weekprior to the surgical procedure. Approximately 1 week prior to the clampexperiment, Tygon catheters are inserted under halothane anaesthesiainto the jugular vein (for infusion) and the carotid artery (for bloodsampling) and exteriorised and fixed on the back of the neck. The ratsare given Streptocilin vet. (Boehringer Ingelheim; 0.15 ml/rat, i.m.)post-surgically and placed in an animal care unit (25° C.) during therecovery period. In order to obtain analgesia, Anorphin (0.06 mg/rat,s.c.) is administered during anaesthesia and Rimadyl (1.5 mg/kg, s.c.)is administered after full recovery from the anaesthesia (2-3 h) andagain once daily for 2 days.

At 7 am on the experimental day overnight fasted (from 3 μm the previousday) rats are weighed and connected to the sampling syringes andinfusion system (Harvard 22 Basic pumps, Harvard, and PerfectumHypodermic glass syringe, Aldrich) and then placed into individual clampcages where they rest for ca. 45 min before start of experiment. Therats are able to move freely on their usual bedding during the entireexperiment and have free access to drinking water. After a 30 min basalperiod during which plasma glucose levels were measured at 10 minintervals, the insulin derivative to be tested and human insulin (onedose level per rat, n=6-7 per dose level) are infused (i.v.) at aconstant rate for 300 min. Optionally a priming bolus infusion of theinsulin derivative to be tested is administered in order to reachimmediate steady state levels in plasma. The dose of the priming bolusinfusion can be calculated based on clearance data obtained from i.v.bolus pharmacokinetics by a pharmacokinetician skilled in the art.Plasma glucose levels are measured at 10 min intervals throughout andinfusion of 20% aqueous glucose is adjusted accordingly in order tomaintain euglyceamia. Samples of re-suspended erythrocytes are pooledfrom each rat and returned in about ½ ml volumes via the carotidcatheter.

On each experimental day, samples of the solutions of the individualinsulin derivatives to be tested and the human insulin solution aretaken before and at the end of the clamp experiments and theconcentrations of the peptides are confirmed by HPLC. Plasmaconcentrations of rat insulin and C-peptide as well as of the insulinderivative to be tested and human insulin are measured at relevant timepoints before and at the end of the studies. Rats are killed at the endof experiment using a pentobarbital overdose.

SEQUENCE LISTS

SEQ ID Nos. 5-11 are the sequences for the A chains present in thecompounds of this invention shown in the above specific examples and SEQID Nos. 12-29 are the sequences for the B chains present in thecompounds of this invention shown in the above specific examples.

What is claimed is:
 1. An acylated protease stabilized insulin, whereinthe acyl moiety is attached to a lysine residue at position B29 of A14E,B16H, B25H, B29K, desB30 human insulin and the acyl moiety is selectedfrom the group consisting of:


2. An acylated protease stabilized insulin which is selected from thegroup consisting of A14E, B16H, B25H,B29K(N^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E,B16H, B25H, B29K(N^(∈)Hexadecanedioyl-γGlu), desB30 human insulin; A14E,B16H, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-γGlu-γGlu), desB30 humaninsulin; A14E, B16H, B25H, B29K(N^(∈)Octadecanedioyl-γGlu-γGlu), desB30human insulin; A14E, B16H, B25H, B29K(N(eps)Eicosanedioyl-γGlu-OEG-OEG),desB30 human insulin; A14E, B25H, B16H, B29K(N^(∈)Octadecanedioyl-γGlu),desB30 human insulin; A14E, B16H, B25H,B29K(N^(∈)Octadecanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]acetyl),desB30 human insulin; and A14E, B16H, B25H,B29K(N^(∈)Eicosanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]acetyl),desB30 human insulin, wherein OEG is NH₂(CH₂)₂O(CH₂)₂OCH₂CO₂H and γGluis gamma glutamic acid.
 3. The acylated protease stabilized insulinaccording to claim 2 which is A14E, B16H, B25H,B29K(NN^(∈)Octadecanedioyl-γGlu-OEG-OEG), desB30 human insulin.
 4. Theacylated protease stabilized insulin according to claim 2 which is A14E,B16H, B25H, B29K(NN^(∈)Hexadecanedioyl-γGlu), desB30 human insulin. 5.The acylated protease stabilized insulin according to claim 2 which isA14E, B16H, B25H, B29K(NN^(∈)Octadecanedioyl-γGlu-γGlu-γGlu), desB30human insulin.
 6. The acylated protease stabilized insulin according toclaim 2 which is A14E, B16H, B25H,B29K(NN^(∈)Octadecanedioyl-γGlu-γGlu), desB30 human insulin.
 7. Theacylated protease stabilized insulin according to claim 2 which is A14E,B16H, B25H, B29K(N(eps)Eicosanedioyl-γGlu-OEG-OEG), desB30 humaninsulin.
 8. The acylated protease stabilized insulin according to claim2 which is A14E, B25H, B16H, B29K(NN^(∈)Octadecanedioyl-γGlu), desB30human insulin.
 9. The acylated protease stabilized insulin according toclaim 2 which is A14E, B16H, B25H,B29K(NN^(∈)Octadecanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]acetyl),desB30 human insulin.
 10. The acylated protease stabilized insulinaccording to claim 2 which is A14E, B16H, B25H,B29K(N^(∈)Eicosanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]acetyl),desB30 human insulin.
 11. A pharmaceutical composition comprising anacylated protease stabilized insulin according to claim 2 in apharmaceutically acceptable carrier or excipient.
 12. A method fortreating a subject with type 1 or type 2 diabetes, said methodcomprising administering to the subject a therapeutically effectiveamount of the pharmaceutical composition of claim
 11. 13. Apharmaceutical composition comprising an acylated protease stabilizedinsulin according to claim 1 in a pharmaceutically acceptable carrier orexcipient.
 14. A method for treating a subject with type 1 or type 2diabetes, said method comprising administering to the subject atherapeutically effective amount of the pharmaceutical composition ofclaim 13.